Development and Comprehensive Benchmark of a High-Quality AMBER-Consistent Small Molecule Force Field with Broad Chemical Space Coverage for Molecular Modeling and Free Energy Calculation.

Biomolecular simulations have become an essential tool in contemporary drug discovery, and molecular mechanics force fields (FFs) constitute its cornerstone. Developing a high quality and broad coverage general FF is a significant undertaking that requires substantial expert knowledge and computing resources, which is beyond the scope of general practitioners. Existing FFs originate from only a limited number of groups and organizations, and they either suffer from limited numbers of training sets, lower than desired quality because of oversimplified representations, or are costly for the molecular modeling community to access. To address these issues, in this work, we developed an AMBER-consistent small molecule FF with extensive chemical space coverage, and we provide Open Access parameters for the entire modeling community. To validate our FF, we carried out benchmarks of quantum mechanics (QM)/molecular mechanics conformer comparison and free energy perturbation calculations on several benchmark data sets. Our FF achieves a higher level of performance at reproducing QM energies and geometries than two popular open-source FFs, OpenFF2 and GAFF2. In relative binding free energy calculations for 31 protein-ligand data sets, comprising 1079 pairs of ligands, the new FF achieves an overall root-mean-square error of 1.19 kcal/mol for ΔΔG and 0.92 kcal/mol for ΔG on a subset of 463 ligands without bespoke fitting to the data sets. The results are on par with those of the leading commercial series of OPLS FFs.

Metallophosphoesterase 1, a novel candidate gene in hepatocellular carcinoma malignancy and recurrence.

BACKGROUND: There is an unmet need to identify novel mechanism-based prognostic genes associated with hepatocellular carcinoma (HCC) recurrence that can predict patient outcomes and provide therapeutic targets. This study aims to identify potential novel driver genes and mutations in HCC. METHODS: Single nucleotide variations (SNVs) contributing to HCC recurrence were identified using whole-exome sequencing of 5 DNA samples extracted from a single HCC patient with HBV-induced cirrhosis. SNVs were verified in primary HCC (n = 87), recurrent HCC (n = 34), and benign liver disease with cirrhosis tissues (n = 43). A candidate gene was identified, and its association and function in HCC development and recurrence were examined. RESULTS: 177 SNVs were identified and 70 SNVs were verified. A MPPE1 missense mutation on chr18_11897016 was the most frequent mutation (16.5%) in primary and recurrent HCC tissues, occurring with a higher frequency in recurrent HCC than primary HCC or benign liver tumor tissues. The MPPE1 mutation was significantly associated with HCC recurrence (P = .003), TNM stage (P = .002), and Child-Pugh classification (P = .039), and was an independent risk factor for HCC recurrence (HR = 1.969; 95%CI = 1.043-3.714, P = .037). Analysis of publically available data deposited in the GEO and TCGA showed MPPE1 expression levels were significantly increased in HCC tumor samples compared to adjacent nontumor tissues. The knockdown of MPPE1 in HCC cell lines significantly inhibited cell proliferation, migration and invasion, induced cell cycle arrest and apoptosis in vitro, and inhibited xenograft tumor growth in nude mice in vivo (P < .05). CONCLUSIONS: MPPE1 is a novel gene associated with HCC malignancy and recurrence.

Cognitive effects of electro-acupuncture and pregabalin in a trigeminal neuralgia rat model induced by cobra venom.

OBJECTIVE: The objective of this study was to investigate the effects of electro-acupuncture (EA) and pregabalin on cognition impairment induced by chronic trigeminal neuralgia (TN) in rats. DESIGN: Controlled animal study. SETTING: Department of Anesthesiology, Pain Medicine and Critical Care Medicine, Aviation General Hospital of China Medical University. SUBJECTS: Forty adult male Sprague Dawley rats. METHODS: Rats were randomly divided into four groups. The TN model was induced by administration of cobra venom to the left infraorbital nerve. On postoperative day 14, either EA or pregabalin was administered, free behavioral activities were observed. Spatial learning and memory abilities were determined in the Morris water maze. The ultrastructural alterations of the Gasserian ganglion, medulla oblongata and hippocampus were examined by electron microscopy. The changes on long-term potentiation were investigated. RESULTS: After treatment, the exploratory behavior increased and the grooming behavior decreased (P<0.05) for the EA group and pregabalin group compared with the cobra venom group; moreover, demyelination of neurons in Gasserian ganglion and medulla oblongata was reversed. The number of platform site crossings, the average percentages of time in the target quadrant and the field excitatory postsynaptic potential slopes increased (P<0.05) in the EA group compared to the cobra venom group. However, the pregabalin group showed no differences compared to the cobra venom group (P>0.05). Vacuolar degeneration in the hippocampal neurons was mild in the EA group, while it was severe in the pregabalin group. CONCLUSION: EA and pregabalin could alleviate TN induced by cobra venom. EA could also inhibit the cognition deficit induced by TN, while pregabalin could not.

Analgesic Effects and Neuropathology Changes of Electroacupuncture on Curing a Rat Model of Brachial Plexus Neuralgia Induced by Cobra Venom.

BACKGROUND: Electroacupuncture (EA) is widely applied to treat neuropathic pain. Brachial plexus neuralgia (BPN) is a common form of chronic persistent pain. Few studies have evaluated the analgesic effects and mechanism of EA using the novel animal model of BPN. OBJECTIVE: To observe the curative effects of repeated EA on curing BPN induced by administration of cobra venom to the lower trunk of the right brachial plexus. STUDY DESIGN: Controlled animal study. SETTING: Department of Anesthesiology, Pain Medicine & Critical Care Medicine, Aviation General Hospital of China Medical University. METHODS: Sixty-six adult male Sprague-Dawley rats were equally and randomly divided into the following groups: normal control (NC), brachial plexus neuralgia (BPN), BPN with sham EA stimulation, BPN with EA stimulation starting on postoperative day 1 (EA1), and BPN with EA stimulation starting on postoperative day 12 (EA12). The BPN model was established by administration of cobra venom to the lower trunk of the right brachial plexus. On postoperative day 1 or day 12, EA (constant aquare wave, 2 Hz and 100 Hz alternating frequencies, intensities ranging from 1 - 1.5 - 2 mA) was applied to the right "Shousanli" (LI10) and "Quchi" (LI11) acupoints for 30 minutes, once every other day for 12 times in both groups. Mechanical withdrawal thresholds (MWT) were tested with von Frey filaments. Video recordings were conducted to analyze the spontaneous exploratory behaviors. Moreover, the organizational and structural alterations of the right brachial plexus and cervical cord (C8-T1) were examined via light and electron microscopy. RESULTS: Following the production of the BPN model, the MWT of both ipsilateral and contralateral paws demonstrated a profound decrease (P < 0.05). But after EA interventions, the MWT showed a significant increase (P < 0.05). In comparison to the EA12 group, the analgesic effects of the EA1 group were more significant, and similar results were observed in exploratory behaviors. However, grooming behaviors did not demonstrate significant differences. Meanwhile, on day 12 after surgery it was observed under light microscopy that the inflammatory response in the right brachial plexus and cervical cord (C8-T1) were significantly attenuated after EA stimulation. Furthermore, the demyelination of the brachial plexus and cervical cord (C8-T1) were also reversed. LIMITATIONS: Limitations include the fact that there was demyelination of the cervical cord (C8-T1) in the control group because of inappropriate manipulation. CONCLUSION: Repeated EA contributes significant analgesic effects in the treatment of BPN.

Rat Model of Trigeminal Neuralgia Using Cobra Venom Mimics the Electron Microscopy, Behavioral, and Anticonvulsant Drug Responses Seen in Patients.

BACKGROUND: A new animal model of trigeminal neuralgia produced by injecting cobra venom into the infraorbital nerve (ION) trunk in rats had been developed. We tested and extended the model by observing the ultrastructural alterations of neurons and ameliorative effect of pregabalin in cobra venom-induced pain behaviors of rats. OBJECTIVES: The goal of this study was to prove the feasibility of the cobra venom-induced model of trigeminal neuralgia and to demonstrate the demyelination change of ION and medulla oblongata is the major pathological change of trigeminal neuralgia. STUDY DESIGN: An experimental study. SETTING: Department of Anesthesiology, Pain Medicine, and Critical Care Medicine, Aviation General Hospital of China Medical University. METHODS: Experiments were carried out on male Sprague-Dawley rats. Video recordings were taken after the cobra venom injection and pregabalin administration. Ultrastructural alterations of ION and medulla oblongata were observed at the electron microscopic level. We also observed alteration in pain behaviors by analysis of video recordings. RESULTS: Compared to the preoperative and sham-operated group rats, experimental group rats exhibited significant changes in exploratory, resting, face-grooming, and head-shake behaviors on 3, 7, 14 days after operation (P < 0.01). The demyelination changes of ION and medulla oblongata were evident after administration of cobra venom to the ION. Compared to the pre-treated (no pregabalin administration) and control group rats, pregabalin group rats showed profound changes in exploratory, resting, face-grooming, and head-shake behaviors throughout the 14 day study period after treatment with drugs (P < 0.01). LIMITATIONS: Ultrastructural alterations of ION and medulla oblongata were not examined after the treatment with pregabalin. CONCLUSIONS: Video recordings of free behaviors and pregabalin application prove the feasibility of the rat model of trigeminal neuralgia. The results of electron micrographs are consistent with a previous study in humans showing that the demyelination change of axons is the major pathological change of trigeminal neuralgia. The central demyelination alterations may explain why the mechanical threshold was found to be decreased on the non-operated side of experimental animals.

A New Animal Model of Brachial Plexus Neuralgia Produced by Injection of Cobra Venom into the Lower Trunk in the Rat.

BACKGROUND: To establish a new animal model for the study of neuropathic pain developed by administration of cobra venom to the brachial plexus (BP) lower trunk. METHODS: Fifty-eight adult male Sprague-Dawley rats were randomly divided into 5 groups. Under pentobarbital sodium anesthesia, cobra venom was injected into the lower trunk or sham operation was performed in the animals. On postoperative day 1 and day 12, pregabalin was administered intragastricly at 30 mg/kg in two groups. Mechanical withdrawal thresholds (MWT) were tested with von Frey filaments. Video recordings were used to analyze the spontaneous behaviors. Meanwhile, our model was confirmed by observing ultrastructural alterations of the BP and cervical cord (C8-T1) via electron microscope examination. RESULTS: In comparison to the blank and sham-operated group, cobra venom-treated rats showed a profound decrease in the MWT, exploratory and increase in grooming behaviors (P<0.05). The changes were long-lasting (up to 60 days), in both ipsilateral and contralateral paws. Furthermore, it was observed under microscopic examination that the myelin sheath was demyelinated in the BP and cervical cord (C8-T1) after injection of cobra venom to the lower trunk. Pregabalin group rats showed changes in MWT and spontaneous behaviors after pregabalin treatment at postoperative day 1 (P>0.05), compared with the control and sham-operated groups. In pregabalin test POD12 group, the decreased MWT and the increased grooming behavior were improved at 20 days after operation. However, pregabalin had no effect on exploratory activity. Results indicate that pregabalin effectively attenuates mechanical hyperalgesia in acute period. CONCLUSIONS: The cobra venom model can be used as a model to induce neuropathic pain and to enable study of the mechanism and treatment.

Prevalence of neutralizing factors against adeno-associated virus types 2 in age-related macular degeneration and polypoidal choroidal vasculopathy.

Adeno-associated virus type 2 (AAV2) mediated gene therapy providing a potential treatment in the eye. However, immune responses can limit virally mediated gene transfer and therapy. To assess preexisting AAV2 neutralizing factors (NF) titers in peripheral blood and the vitreous in patients with age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV). 130 subjects were enrolled: 50 with neovascular AMD, 30 with PCV, and 50 controls. The serum and the vitreous were obtained for AAV2 NF assay. We found AAV2 NF are present in all of AMD, PCV patients and controls we tested. There were no significant differences in prevalence of NAb in serum between AMD, PCV and controls (P=0.999). There was no correlation between NF in serum and in vitreous (P>0.05), and NF in vitreous was significantly less than in serum. Our results for the first time showed in Chinese population, NF against AAV2 was present in serum of all the patients with AMD or PCV and controls, and there were no significant differences among these groups. Therefore, it demonstrated there were no correlations between AAV2 NF titer and these diseases. We found NF in vitreous was considerably less than in serum in all groups. We also found no direct correlation between NF in vitreous and in serum suggesting serum antibody levels may not be used to predict their counterparts in the vitreous. Our results will provide crucial information for future clinical studies in the development of new therapies based on AAV2 mediated gene delivery in the eye.

Direct comparison of the potency of human mesenchymal stem cells derived from amnion tissue, bone marrow and adipose tissue at inducing dermal fibroblast responses to cutaneous wounds.

Although transplantation of human mesenchymal stem cells (MSCs) derived from amnion (hAMSCs), bone marrow (hBMSCs) and adipose tissues (hADSCs) has been shown to aid in the repair of cutaneous wounds in mouse models, little information is available regarding the relative efficacy of MSCs from different sources. In this study, we compared their therapeutic potentials by transplanting equal numbers of hAMSCs, hBMSCs or hADSCs in a mouse model of cutaneous wounds. The results suggested that an hADSC injection has the most pronounced effect on wound closure. Histological evaluation showed enhanced re-epithelialization in the hADSC group compared with the hBMSC and hAMSC groups. Although there was a slight improvement in wound healing in the hAMSC and hBMSC groups, the differences between the groups were statistically insignificant. In a trans-well coculture model, wound healing migration and transwell migration assays showed that hADSCs were superior to hAMSCs and hBMSCs at promoting human dermal fibroblast (hDF) migration. However, there was no significant difference in fibroblast proliferation between the hAMSC, hBMSC and hADSC groups, as measured by WST assay. Our results also indicated that hDFs cocultured with hADSCs for 48 h significantly upregulated their mRNA expression of the cytokine vascular endothelial growth factor, basic fibroblast growth factor, keratinocyte growth factor and transforming growth factor-ß and increased the mRNA and protein levels of type I collagen. Collectively, these data suggest that hADSCs are a potential source of MSCs for therapeutic healing in cutaneous wounds in terms of efficacy, accessibility and availability.

Alemtuzumab induction of intracellular signaling and apoptosis in malignant B lymphocytes.

The molecular changes induced by alemtuzumab following binding of CD52 on B tumor cells were investigated. Alemtuzumab alone had no detectable impact on cell signaling but cross-linking of alemtuzumab on the surface of B tumor lines with anti-human Fc antibodies induced a transient Ca(2+) flux followed by phosphorylation of several kinases involved in stress and survival pathways, and expression of associated proteins including TNF-α. Cross-linking of alemtuzumab also induced capping and caspase-dependent apoptosis of the tumor lines. When using primary cells from B-CLL patients, alemtuzumab alone was capable of inducing protein phosphorylation and apoptosis through the cross-linking of alemtuzumab by FcγRIIb receptors on B-CLL cells. Apoptosis was prevented by blocking of FcγRIIb receptors with anti-CD32 antibody. Overall, our results indicate that cross-linking of alemtuzumab on B tumor cells can occur naturally through Fc receptor interaction and leads to the activation of specific cellular pathways and induction of apoptosis.

The chemosensitizing activity of inhibitors of glucosylceramide synthase is mediated primarily through modulation of P-gp function.

Glucosylceramide synthase (GCS) is a key enzyme engaged in the biosynthesis of glycosphingolipids and in regulating ceramide metabolism. Studies exploring alterations in GCS activity suggest that the glycolase may have a role in chemosensitizing tumor cells to various cancer drugs. The chemosensitizing effect of inhibitors of GCS (e.g. PDMP and selected analogues) has been observed with a variety of tumor cells leading to the proposal that the sensitizing activity of GCS inhibitors is primarily through increases in intracellular ceramide leading to induction of apoptosis. The current study examined the chemosensitizing activity of the novel GCS inhibitor, Genz-123346 in cell culture. Exposure of cells to Genz-123346 and to other GCS inhibitors at non-toxic concentrations can enhance the killing of tumor cells by cytotoxic anti-cancer agents. This activity was unrelated to lowering intracellular glycosphingolipid levels. Genz-123346 and a few other GCS inhibitors are substrates for multi-drug resistance efflux pumps such as P-gp (ABCB1, gP-170). In cell lines selected to over-express P-gp or which endogenously express P-gp, chemosensitization by Genz-123346 was primarily due to the effects on P-gp function. RNA interference studies using siRNA or shRNA confirmed that lowering GCS expression in tumor cells did not affect their responsiveness to commonly used cytotoxic drugs.

Knockdown of human deubiquitinase PSMD14 induces cell cycle arrest and senescence.

The PSMD14 (POH1, also known as Rpn11/MPR1/S13/CepP1) protein within the 19S complex (19S cap; PA700) is responsible for substrate deubiquitination during proteasomal degradation. The role of PSMD14 in cell proliferation and senescence was explored using siRNA knockdown in carcinoma cell lines. Our results reveal that down-regulation of PSMD14 by siRNA transfection had a considerable impact on cell viability causing cell arrest in the G0-G1 phase, ultimately leading to senescence. The molecular events associated with decreased cell proliferation, cell cycle arrest and senescence include down-regulation of cyclin B1-CDK1-CDC25C, down-regulation of cyclin D1 and up-regulation of p21(/Cip) and p27(/Kip1). Most notably, phosphorylation of the retinoblastoma protein was markedly reduced in PSMD14 knockdown cells. A comparative study with PSMB5, a subunit of the 20S proteasome, revealed that PSMB5 and PSMD14 have different effects on cell cycle, senescence and associated molecular events. These data support the view that the 19S and 20S subunits of the proteasome have distinct biological functions and imply that targeting 19S and 20S would have distinct molecular consequences on tumor cells.

Netrin-4 regulates angiogenic responses and tumor cell growth.

Netrin-4 is a 628 amino acid basement membrane component that promotes neurite elongation at low concentrations but inhibits neurite extension at high concentrations. There is a growing body of literature suggesting that several molecules, including netrins, are regulators of both neuronal and vascular growth. It is believed that molecules that guide neural growth and development are also involved in regulating morphogenesis of the vascular tree. Further, netrins have recently been implicated in controlling epithelial cell branching morphogenesis in the breast, lung and pancreas. Characterization of purified netrin-4 in in vitro angiogenesis assays demonstrated that netrin-4 markedly inhibits HMVEC migration and tube formation. Moreover, netrin-4 inhibits proliferation of a variety of human tumor cells in vitro. Netrin-4 has only modest effects on proliferation of endothelial and other non-transformed cells. Netrin-4 treatment results in phosphorylation changes of proteins that are known to control cell growth. Specifically, Phospho-Akt-1, Phospho-Jnk-2, and Phospho-c-Jun are reduced in tumor cells that have been treated with netrin-4. Together, these data suggest a potential role for netrin-4 in regulating tumor growth.

Regional mutagenesis of the gene encoding the phage Mu late gene activator C identifies two separate regions important for DNA binding.

Lytic development of bacteriophage Mu is controlled by a regulatory cascade and involves three phases of transcription: early, middle and late. Late transcription requires the host RNA polymerase holoenzyme and a 16.5-kDa Mu-encoded activator protein C. Consistent with these requirements, the four late promoters P(lys), P(I), P(P) and P(mom) have recognizable -10 hexamers but lack typical -35 hexamers. The C protein binds to a 16-bp imperfect dyad-symmetrical sequence element centered at -43.5 and overlapping the -35 region. Based on the crystal structure of the closely related Mor protein, the activator of Mu middle transcription, we predict that two regions of C are involved in DNA binding: a helix-turn-helix region and a beta-strand region linking the dimerization and helix-turn-helix domains. To test this hypothesis, we carried out mutagenesis of the corresponding regions of the C gene by degenerate oligonucleotide-directed PCR and screened the resulting mutants for their ability to activate a P(lys)-galK fusion. Analysis of the mutant proteins by gel mobility shift, beta-galactosidase and polyacrylamide gel electrophoresis assays identified a number of amino acid residues important for C DNA binding in both regions.

Regulation of long chain unsaturated fatty acid synthesis in yeast.

Saccharomyces cerevisiae forms monounsaturated fatty acids using the ER membrane-bound Delta-9 fatty acid desaturase, Ole1p, an enzyme system that forms a double bond in saturated fatty acyl CoA substrates. Ole1p is a chimeric protein consisting of an amino terminal desaturase domain fused to cytochrome b5. It catalyzes the formation of the double bond through an oxygen-dependent mechanism that requires reducing equivalents from NADH. These are transferred to the enzyme via NADH cytochrome b5 reductase to the Ole1p cytochrome b5 domain and then to the diiron-oxo catalytic center of the enzyme. The control of OLE1 gene expression appears to mediated through the ER membrane proteins Spt23p and Mga2p. N-terminal fragments of these proteins are released by an ubiquitin/proteasome mediated proteolysis system and translocated to the nucleus where they appear to act as transcription coactivators of OLE1. OLE1 is regulated through Spt23p and Mga2p by multiple systems that control its transcription and mRNA stability in response to diverse stimuli that include nutrient fatty acids, carbon source, metal ions and the availability of oxygen.

Alterations in vascular gene expression in invasive breast carcinoma.

The molecular signature that defines tumor microvasculature will likely provide clues as to how vascular-dependent tumor proliferation is regulated. Using purified endothelial cells, we generated a database of gene expression changes accompanying vascular proliferation in invasive breast cancer. In contrast to normal mammary vasculature, invasive breast cancer vasculature expresses extracellular matrix and surface proteins characteristic of proliferating and migrating endothelial cells. We define and validate the up-regulated expression of VE-cadherin and osteonectin in breast tumor vasculature. In contrast to other tumor types, invasive breast cancer vasculature induced a high expression level of specific transcription factors, including SNAIL1 and HEYL, that may drive gene expression changes necessary for breast tumor neovascularization. We demonstrate the expression of HEYL in tumor endothelial cells and additionally establish the ability of HEYL to both induce proliferation and attenuate programmed cell death of primary endothelial cells in vitro. We also establish that an additional intracellular protein and previously defined metastasis-associated gene, PRL3, appears to be expressed predominately in the vasculature of invasive breast cancers and is able to enhance the migration of endothelial cells in vitro. Together, our results provide unique insights into vascular regulation in breast tumors and suggest specific roles for genes in driving tumor angiogenesis.

Vascular gene expression in nonneoplastic and malignant brain.

Malignant gliomas are uniformly lethal tumors whose morbidity is mediated in large part by the angiogenic response of the brain to the invading tumor. This profound angiogenic response leads to aggressive tumor invasion and destruction of surrounding brain tissue as well as blood-brain barrier breakdown and life-threatening cerebral edema. To investigate the molecular mechanisms governing the proliferation of abnormal microvasculature in malignant brain tumor patients, we have undertaken a cell-specific transcriptome analysis from surgically harvested nonneoplastic and tumor-associated endothelial cells. SAGE-derived endothelial cell gene expression patterns from glioma and nonneoplastic brain tissue reveal distinct gene expression patterns and consistent up-regulation of certain glioma endothelial marker genes across patient samples. We define the G-protein-coupled receptor RDC1 as a tumor endothelial marker whose expression is distinctly induced in tumor endothelial cells of both brain and peripheral vasculature. Further, we demonstrate that the glioma-induced gene, PV1, shows expression both restricted to endothelial cells and coincident with endothelial cell tube formation. As PV1 provides a framework for endothelial cell caveolar diaphragms, this protein may serve to enhance glioma-induced disruption of the blood-brain barrier and transendothelial exchange. Additional characterization of this extensive brain endothelial cell gene expression database will provide unique molecular insights into vascular gene expression.

Gene expression profiling in a renal cell carcinoma cell line: dissecting VHL and hypoxia-dependent pathways.

The von Hippel-Lindau tumor suppressor, pVHL, is a key player in one of the best characterized hypoxia signaling pathways, the VHL-hypoxia-inducible factor (VHL-HIF) pathway. To better understand the role of VHL in the hypoxia signaling pathways of tumor cells, we used serial analysis of gene expression (SAGE) to investigate hypoxia-regulated gene expression in renal carcinoma cells (786-0), with and without VHL. The gene expression profiles of the cancer cells were compared to SAGE profiles from normal renal proximal tubule cells grown under both normoxia and hypoxia. The data suggest that the role of VHL as a tumor suppressor may be more complex than previously thought. Further, the data reveal that renal carcinoma cells have evolved an alternative hypoxia signaling pathway(s) compared with normal renal cells. These alternative hypoxia pathways demonstrate VHL-dependent and VHL-independent regulation. The genes involved in such pathways include those with potential importance in the physiological and pathological regulation of tumor growth and angiogenesis. Some of the genes identified as showing overexpression in the cancer cells, particularly those encoding secreted or membrane-bound proteins, could be potential biomarkers for tumors or targets for rational therapeutics that are dependent on VHL status.

Mga2p processing by hypoxia and unsaturated fatty acids in Saccharomyces cerevisiae: impact on LORE-dependent gene expression.

In Saccharomyces cerevisiae, OLE1 encodes a delta9 fatty acid desaturase, an enzyme that plays a critical role in maintaining the correct ratio of saturated to monounsaturated fatty acids in the cell membrane. Previous studies have demonstrated that (i) OLE1 expression is repressed by unsaturated fatty acids (UFAs) and induced by low oxygen tension, (ii) a component of this regulation is mediated through the same low oxygen response element (LORE) in the OLE1 promoter, and (iii) Mga2p is involved in LORE-dependent hypoxic induction of OLE1. We now report that LORE-CYC1 basal promoter-lacZ fusion reporter assays demonstrate that UFAs repress the reporter expression under hypoxic conditions in a dose-dependent manner via LORE. Electrophoretic mobility shift assays show that UFAs repress the hypoxia-induced complex formation with LORE. Studies with a construct encoding a truncated form of Mga2p support the hypothesis that both hypoxia and UFA signals affect the processing of Mga2p and the UFA repression of OLE1 hypoxic induction is mediated through Mga2p. Data from Western blot assays provide evidence that under normoxic conditions, Mga2p processing produces approximately equimolar levels of the membrane-bound and processed forms and is unaffected by UFAs. Hypoxic induction of OLE1, however, is associated with increased processing of the protein, resulting in an approximately fivefold increase in the soluble active form that is counteracted by exposure of the cells to unsaturated fatty acids. Data from this study suggest that the Mga2p-LORE interaction plays an important role in OLE1 expression under both normoxic and hypoxic conditions.