Structure of an integral membrane sterol reductase from Methylomicrobium alcaliphilum.

Sterols are essential biological molecules in the majority of life forms. Sterol reductases including Δ(14)-sterol reductase (C14SR, also known as TM7SF2), 7-dehydrocholesterol reductase (DHCR7) and 24-dehydrocholesterol reductase (DHCR24) reduce specific carbon-carbon double bonds of the sterol moiety using a reducing cofactor during sterol biosynthesis. Lamin B receptor (LBR), an integral inner nuclear membrane protein, also contains a functional C14SR domain. Here we report the crystal structure of a Δ(14)-sterol reductase (MaSR1) from the methanotrophic bacterium Methylomicrobium alcaliphilum 20Z (a homologue of human C14SR, LBR and DHCR7) with the cofactor NADPH. The enzyme contains ten transmembrane segments (TM1-10). Its catalytic domain comprises the carboxy-terminal half (containing TM6-10) and envelops two interconnected pockets, one of which faces the cytoplasm and houses NADPH, while the other one is accessible from the lipid bilayer. Comparison with a soluble steroid 5ß-reductase structure suggests that the reducing end of NADPH meets the sterol substrate at the juncture of the two pockets. A sterol reductase activity assay proves that MaSR1 can reduce the double bond of a cholesterol biosynthetic intermediate, demonstrating functional conservation to human C14SR. Therefore, our structure as a prototype of integral membrane sterol reductases provides molecular insight into mutations in DHCR7 and LBR for inborn human diseases.

Optimization of DamID for use in primary cultures of mouse hepatocytes.

DamID, a method to identify DNA associating with a particular protein, was originally developed for use in immortalized tissue culture lines. The power of this technique has led to its adaptation for a number of additional systems. Here we report adaptations for its use in primary cells isolated from rodents with emphasis on the challenges this presents. Specifically, we present several modifications that allow the method to be performed in mouse acutely isolated primary hepatocytes while seemingly maintaining tissue genome architecture. We also describe the downstream bioinformatic analysis necessary to identify LADs and discuss some of the parameters and their effects with regards to the sensitivity of the method.

Selected cholesterol biosynthesis inhibitors produce accumulation of the intermediate FF-MAS that targets nucleus and activates LXRα in HepG2 cells.

Sterol intermediates of the cholesterol biosynthetic pathway have drawn attention for novel biological activities. Follicular fluid meiosis activating sterol (FF-MAS) is a LXRα ligand and a potential modulator of physiologic processes regulated by nuclear receptors, such as lipid homeostasis and cell proliferation. In this work, we established a model to selectively accumulate FF-MAS in HepG2 cells, by using a combination of the inhibitors AY9944 and 17-hydroxyprogesterone to block C14-sterol reductases and the downstream C4-demethylase complex. We investigated the effects produced by altered levels of cholesterol biosynthesis intermediates, in order to dissect their influence on LXRα signaling. In particular, endogenously accumulated FF-MAS was able to modulate the expression of key genes in cholesterol metabolism, to activate LXRα nuclear signaling resulting in increased lipogenesis, and to inhibit HepG2 cells proliferation. Moreover, a fluorescent ester derivative of FF-MAS localized in nuclear lipid droplets, suggesting a role for these organelles in the storage of signaling lipids interacting with nuclear partners.

A novel killer protein from Pichia kluyveri isolated from an Algerian soil: purification and characterization of its in vitro activity against food and beverage spoilage yeasts.

A novel killer protein (Pkkp) secreted by a Pichia kluyveri strain isolated from an Algerian soil was active against food and beverage spoilage yeasts of the genera Dekkera, Kluyveromyces, Pichia, Saccharomyces, Torulaspora, Wickerhamomyces and Zygosaccharomyces. After purification by gel filtration chromatography Pkkp revealed an apparent molecular mass of 54 kDa with SDS-PAGE. Minimum inhibitory concentrations (MICs) of purified Pkkp exhibited a high in vitro activity against Dekkera bruxellensis (MICs from 64,000- to 256,000-fold lower than that exhibited by potassium metabisulphite) and Saccharomyces cerevisiae (MICs from 32,000- to 64,000- fold lower than potassium sorbate). No in vitro synergistic interactions (calculated by FIC index - Σ FIC) were observed when Pkkp was used in combination with potassium metabisulphite, potassium sorbate, or ethanol. Pkkp exhibited a dose-response effect against D. bruxellensis and S. cerevisiae in a low-alcoholic drink and fruit juice, respectively. The results of the present study suggest that Pkkp could be proposed as a novel food-grade compound useful for the control of food and beverage spoilage yeasts.

The energy blockers 3-bromopyruvate and lonidamine: effects on bioenergetics of brain mitochondria.

Tumor cells favor abnormal energy production via aerobic glycolysis and show resistance to apoptosis, suggesting the involvement of mitochondrial dysfunction. The differences between normal and cancer cells in their energy metabolism provide a biochemical basis for developing new therapeutic strategies. The energy blocker 3-bromopyruvate (3BP) can eradicate liver cancer in animals without associated toxicity, and is a potent anticancer towards glioblastoma cells. Since mitochondria are 3BP targets, in this work the effects of 3BP on the bioenergetics of normal rat brain mitochondria were investigated in vitro, in comparison with the anticancer agent lonidamine (LND). Whereas LND impaired oxygen consumption dependent on any complex of the respiratory chain, 3BP was inhibitory to malate/pyruvate and succinate (Complexes I and II), but preserved respiration from glycerol-3-phosphate and ascorbate (Complex IV). Accordingly, although electron flow along the respiratory chain and ATP levels were decreased by 3BP in malate/pyruvate- and succinate-fed mitochondria, they were not significantly influenced from glycerol-3-phosphate- or ascorbate-fed mitochondria. LND produced a decrease in electron flow from all substrates tested. No ROS were produced from any substrate, with the exception of 3BP-induced H(2)O(2) release from succinate, which suggests an antimycin-like action of 3BP as an inhibitor of Complex III. We can conclude that 3BP does not abolish completely respiration and ATP synthesis in brain mitochondria, and has a limited effect on ROS production, confirming that this drug may have limited harmful effects on normal cells.

Lamin B receptor regulates the growth and maturation of myeloid progenitors via its sterol reductase domain: implications for cholesterol biosynthesis in regulating myelopoiesis.

Lamin B receptor (LBR) is a bifunctional nuclear membrane protein with N-terminal lamin B and chromatin-binding domains plus a C-terminal sterol Δ(14) reductase domain. LBR expression increases during neutrophil differentiation, and deficient expression disrupts neutrophil nuclear lobulation characteristic of Pelger-Huët anomaly. Thus, LBR plays a critical role in regulating myeloid differentiation, but how the two functional domains of LBR support this role is currently unclear. We previously identified abnormal proliferation and deficient functional maturation of promyelocytes (erythroid, myeloid, and lymphoid [EML]-derived promyelocytes) derived from EML-ic/ic cells, a myeloid model of ichthyosis (ic) bone marrow that lacks Lbr expression. In this study, we provide new evidence that cholesterol biosynthesis is important to myeloid cell growth and is supported by the sterol reductase domain of Lbr. Cholesterol biosynthesis inhibitors caused growth inhibition of EML cells that increased in EML-derived promyelocytes, whereas cells lacking Lbr exhibited complete growth arrest at both stages. Lipid production increased during wild-type neutrophil maturation, but ic/ic cells exhibited deficient levels of lipid and cholesterol production. Ectopic expression of a full-length Lbr in EML-ic/ic cells rescued both nuclear lobulation and growth arrest in cholesterol starvation conditions. Lipid production also was rescued, and a deficient respiratory burst was corrected. Expression of just the C-terminal sterol reductase domain of Lbr in ic/ic cells also improved each of these phenotypes. Our data support the conclusion that the sterol Δ(14) reductase domain of LBR plays a critical role in cholesterol biosynthesis and that this process is essential to both myeloid cell growth and functional maturation.

Lysosomal di-N-acetylchitobiase-deficient mouse tissues accumulate Man2GlcNAc2 and Man3GlcNAc2.

Most lysosomal storage diseases are caused by defects in genes encoding for acidic hydrolases. Deficiency of an enzyme involved in the catabolic pathway of N-linked glycans leads to the accumulation of the respective substrate and consequently to the onset of a specific storage disorder. Di-N-acetylchitobiase and core specific α1-6mannosidase represent the only exception. In fact, to date no lysosomal disease has been correlated to the deficiency of these enzymes. We generated di-N-acetylchitobiase-deficient mice by gene targeting of the Ctbs gene in murine embryonic stem cells. Accumulation of Man2GlcNAc2 and Man3GlcNAc2 was evaluated in all analyzed tissues and the tetrasaccharide was detected in urines. Multilamellar inclusion bodies reminiscent of polar lipids were present in epithelia of a scattered subset of proximal tubules in the kidney. Less constantly, enlarged Kupffer cells were observed in liver, filled with phagocytic material resembling partly digested red blood cells. These findings confirm an important role for lysosomal di-N-acetylchitobiase in glycans degradation and suggest that its deficiency could be the cause of a not yet described lysosomal storage disease.

H(2)O(2) disposal in cardiolipin-enriched brain mitochondria is due to increased cytochrome c peroxidase activity.

The mitochondrial electron transport chain is a source of oxygen superoxide anion (O(2)(-)) that is dismutated to H(2)O(2). Although low levels of ROS are physiologically synthesized during respiration, their increase contributes to cell injury. Therefore, an efficient machinery for H(2)O(2) disposal is essential in mitochondria. In this study, the ability of brain mitochondria to acquire cardiolipin (CL), phosphatidylglycerol (PG), and phosphatidylserine (PS) in vitro through a fusion process was exploited to investigate lipid effects on ROS. MTT assay, oxygen consumption, and respiratory ratio indicated that the acquired phospholipids did not alter mitochondrial respiration and O(2)(-) production from succinate. However, in CL-enriched mitochondria, H(2)O(2) levels where 27% and 47% of control in the absence and in the presence of antimycin A, respectively, suggesting an increase in H(2)O(2) elimination. Concomitantly, cytochrome c (cyt c) was released outside mitochondria. Since free oxidized cyt c acquired peroxidase activity towards H(2)O(2) upon interaction with CL in vitro, a contribution of cyt c to H(2)O(2) disposal in mitochondria through CL conferred peroxidase activity is plausible. In this model, the accompanying CL peroxidation should weaken cyt c-CL interactions, favouring the detachment and release of the protein. Neither cyt c peroxidase activity was elicited by PS in vitro, nor cyt c release was observed in PS-enriched mitochondria, although H(2)O(2) levels were significantly decreased, suggesting a cyt c-independent role of PS in ROS metabolism in mitochondria.

Bromopyruvate mediates autophagy and cardiolipin degradation to monolyso-cardiolipin in GL15 glioblastoma cells.

The GL15 glioblastoma cell line undergoes viability loss upon treatment with bromopyruvate. The biochemical mechanisms triggered by the antiglycolytic agent indicate the activation of an autophagic pathway. Acridine orange stains acidic intracellular vesicles already 60 min after bromopyruvate treatment, whereas autophagosomes engulfing electron dense material are well evidenced 18 h later. The autophagic process is accompanied by the expression of the early autophagosomal marker Atg5 and by LC3-II formation, a late biochemical marker associated with autophagosomes. In agreement with the autophagic route activation, the inhibitory and the activator Akt and ERK signaling pathways are depressed and enhanced, respectively. In spite of the energetic collapse suffered by bromopyruvate-treated cells, MALDI-TOF mass spectrometry lipid analysis does not evidence a decrease of the major phospholipids, in accordance with the need of phospholipids for autophagosomal membranes biogenesis. Contrarily, mitochondrial cardiolipin decreases, accompanied by monolyso-cardiolipin formation and complete cytochrome c degradation, events that could target mitochondria to autophagy. However, in our experimental conditions cytochrome c degradation seems to be independent of the autophagic process.

Activation of TM7SF2 promoter by SREBP-2 depends on a new sterol regulatory element, a GC-box, and an inverted CCAAT-box.

TM7SF2 gene encodes 3beta-hydroxysterol Delta(14)-reductase, responsible for the reduction of C14-unsaturated sterols in cholesterol biosynthesis. TM7SF2 gene expression is controlled by cell sterol levels through the SREBP-2. The motifs of TM7SF2 promoter responsible for activation by SREBP-2 have not been characterized. Using electrophoretic mobility shift assays and mutation analysis, we identified a new SRE motif, 60% identical to an inverted SRE-3, able to bind SREBP-2 in vitro and in vivo. Co-transfection of promoter-luciferase reporter constructs in HepG2 cells showed that the binding of SREBP-2 to SRE produced approximately 26-fold promoter activation, whereas mutation of the SRE motif caused a dramatic decrease of transactivation by SREBP-2. The function of additional motifs that bind transcription factors cooperating with SREBP-2 was investigated. An inverted CCAAT-box, that binds nuclear factor Y (NF-Y), cooperates with SREBP-2 in TM7SF2 promoter activation. Deletion of this motif resulted in the loss of promoter induction by sterol starvation in HepG2 cells, as well as a decrease in fold activation by SREBP-2 in co-transfection experiments. Moreover, co-transfection of the promoter with a plasmid expressing dominant negative NF-YA did not permit full activation by SREBP-2. Three GC-boxes (1, 2, 3), known to bind Sp1 transcription factor, were also investigated. The mutagenesis of each of them produced a decrease in SREBP-2-dependent activation, the most powerful being GC-box2. A triple mutagenized promoter construct did not have an additive effect. We conclude that, besides the SRE motif, both the inverted CCAAT-box and GC-box2 are essential for full promoter activation by SREBP-2.

Mitochondrial dysfunction and effect of antiglycolytic bromopyruvic acid in GL15 glioblastoma cells.

Most cancer cells, including GL15 glioblastoma cells, rely on glycolysis for energy supply. The effect of antiglycolytic bromopyruvate on respiratory parameters and viability of GL15 cells was investigated. Bromopyruvate caused Δψ(m) and MTT collapse, ATP decrease, and cell viability loss without involving apoptotic or necrotic pathways. The autophagy marker LC3-II was increased. Δψ(m) decrease was accompanied by reactive oxygen species (ROS) increase and cytochrome c (cyt c) disappearance, suggesting a link between free radical generation and intramitochondrial cyt c degradation. Indeed, the free radical inducer menadione caused a decrease in cyt c that was reversed by N-acetylcysteine. Cyt c is tightly bound to the inner mitochondrial membrane in GL15 cells, which may confer protein peroxidase activity, resulting in auto-oxidation and protein targeting to degradation in the presence of ROS. This process is directed towards impairment of the apoptotic cyt c cascade, although cells are committed to die.

Effect of dietary saturated fatty acids on HNF-4α DNA binding activity and ApoCIII mRNA in sedentary rat liver.

Hind limb-suspended rats represent a sedentary-hyperinsulinemic model with a liver dyslipidemia mainly related to changes in sterol regulatory element-binding protein 1 (SREBP-1) and peroxisome proliferator-activated receptor-α (PPARα) expression and activity. To assess the effects of dietary fatty acids on hepatic lipid homeostasis, the hepatic expression and activity of PPARα, SREBP-1, and hepatocyte nuclear factor-4α (HNF-4α) were investigated in this animal model. In control and sedentary rats, diets enriched with saturated, monounsaturated, and polyunsaturated fatty acids (PUFA) enhanced the expression of the PPARα target genes carnitine palmitoyltransferase 1 and acyl-CoA oxidase, the highest effect being exerted by ω-3. The same diets reduced SREBP-1 mRNA and target lipogenic gene expression, as indicated by the reduction in fatty acid synthase and acetyl-CoA carboxylase mRNA content. Effects were greater in sedentary rat liver than in controls on the same diet. Only the ω-3 enriched diet decreased liver triglyceride content as well as plasma cholesterol and triglyceride levels in sedentary rats. This effect may be mainly related to the enhanced mitochondrial and peroxisomal ß-oxidation genes expression. On the other hand, saturated fatty acid-enriched diet induced an increase in liver triglyceride content and enhanced plasma cholesterol and triglyceride levels, both in control and immobilized rats. This detrimental effect may be ascribed to the induced HNF-4α binding activity on ApoCIII promoter and to the enhanced ApoCIII mRNA levels both in control and in sedentary rat livers. In conclusion, we can speculate that dietary saturated fats, acting at apolipoprotein transcriptional level, may impact on the close relationship existing among high ApoCIII plasma level, dyslipidemia, and atherosclerosis.

Tm7sf2 gene promotes adipocyte differentiation of mouse embryonic fibroblasts and improves insulin sensitivity.

Adipogenesis is a finely orchestrated program involving a transcriptional cascade coordinated by CEBP and PPAR family members and by hormonally induced signaling pathways. Alterations in any of these factors result into impaired formation of fully differentiated adipocytes. Tm7sf2 gene encodes for a Δ(14)-sterol reductase primarily involved in cholesterol biosynthesis. Furthermore, TM7SF2 modulates the expression of the master gene of adipogenesis PPARγ, suggesting a role in the regulation of adipose tissue homeostasis. We investigated the differentiation of Tm7sf2-/- MEFs into adipocytes, compared to Tm7sf2+/+ MEFs. Tm7sf2 expression was increased at late stage of differentiation in wild type cells, while Tm7sf2-/- MEFs exhibited a reduced capacity to differentiate into mature adipocytes. Indeed, Tm7sf2-/- MEFs had lower neutral lipid accumulation and reduced expression of adipogenic regulators. At early stage, the reduction in C/EBPß expression impaired mitotic clonal expansion, which is needed by preadipocytes for adipogenesis induction. At late stage, the expression and activity of C/EBPα and PPARγ were inhibited in Tm7sf2-/- cells, leading to the reduced expression of adipocyte genes like Srebp-1c, Fasn, Scd-1, Adipoq, Fabp4, and Glut4. Loss of the acquisition of adipocyte phenotype was accompanied by a reduction in the levels of Irs1, and phosphorylated Akt and ERK1/2, indicating a blunted insulin signaling in differentiating Tm7sf2-/- cells. Moreover, throughout the differentiation process, increased expression of the antiadipogenic Mmp3 was observed in MEFs lacking Tm7sf2. These findings indicate Tm7sf2 as a novel factor influencing adipocyte differentiation that could be relevant to adipose tissue development and maintenance of metabolic health.

Cytochrome c redox state influences the binding and release of cytochrome c in model membranes and in brain mitochondria.

Cytochrome c (cyt c), a component of the respiratory chain, promotes apoptosis when released into the cytosol. Cyt c anchorage within mitochondria depends on cardiolipin (CL). Detachment and release have been related to CL loss and peroxidation. We report that NaN(3)-dependent complex IV inhibition, accompanied by impairment of respiration, resulted in cyt c release. Contrarily, inhibition of respiration upstream cyt c with complex I and III inhibitors was not accompanied by the release of the protein, despite CL decrease and monolyso-CL increase. No CL changes and H(2)O(2) formation were observed by inhibiting complex IV. In cyt c-CL liposomes, breaching cyt c-CL hydrophilic interactions produced a higher release of the reduced, compared to the oxidized form, suggesting that the hydrophobic component of cyt c-CL binding is prevalent in the oxidized form. Free or liposome-reconstituted cyt c was able to form fatty acid-protein complexes (palmitate < linoleate < oleate) only in its reduced form. We hypothesize that reduced cyt c-fatty acid binding favors the dislocation of the protein from anchoring CL. A mechanism for cyt c release independent of CL peroxidation by H(2)O(2) is feasible. It could weaken the hydrophobic component of cyt c-CL interactions and might function following complex IV inhibition or in oxygen lack, both conditions producing accumulation of reduced cyt c and free fatty acids.

Tm7sf2 Disruption Alters Radial Gene Positioning in Mouse Liver Leading to Metabolic Defects and Diabetes Characteristics.

Tissue-specific patterns of radial genome organization contribute to genome regulation and can be established by nuclear envelope proteins. Studies in this area often use cancer cell lines, and it is unclear how well such systems recapitulate genome organization of primary cells or animal tissues; so, we sought to investigate radial genome organization in primary liver tissue hepatocytes. Here, we have used a NET47/Tm7sf2-/- liver model to show that manipulating one of these nuclear membrane proteins is sufficient to alter tissue-specific gene positioning and expression. Dam-LaminB1 global profiling in primary liver cells shows that nearly all the genes under such positional regulation are related to/important for liver function. Interestingly, Tm7sf2 is a paralog of the HP1-binding nuclear membrane protein LBR that, like Tm7sf2, also has an enzymatic function in sterol reduction. Fmo3 gene/locus radial mislocalization could be rescued with human wild-type, but not TM7SF2 mutants lacking the sterol reductase function. One central pathway affected is the cholesterol synthesis pathway. Within this pathway, both Cyp51 and Msmo1 are under Tm7sf2 positional and expression regulation. Other consequences of the loss of Tm7sf2 included weight gain, insulin sensitivity, and reduced levels of active Akt kinase indicating additional pathways under its regulation, several of which are highlighted by mispositioning genes. This study emphasizes the importance for tissue-specific radial genome organization in tissue function and the value of studying genome organization in animal tissues and primary cells over cell lines.

The efficacy of the anticancer 3-bromopyruvate is potentiated by antimycin and menadione by unbalancing mitochondrial ROS production and disposal in U118 glioblastoma cells.

Metabolic reprogramming of tumour cells sustains cancer progression. Similar to other cancer cells, glioblastoma cells exhibit an increased glycolytic flow, which encourages the use of antiglycolytics as an effective complementary therapy. We used the antiglycolytic 3-bromopyruvate (3BP) as a metabolic modifier to treat U118 glioblastoma cells and investigated the toxic effects and the conditions to increase drug effectiveness at the lowest concentration. Cellular vitality was not affected by 3BP concentrations lower than 40 µM, although p-Akt dephosphorylation, p53 degradation, and ATP reduction occurred already at 30 µM 3BP. ROS generated in mitochondria were enhanced at 30 µM 3BP, possibly by unbalancing their generation and their disposal because of glutathione peroxidase inhibition. ROS triggered JNK and ERK phosphorylation, and cyt c release outside mitochondria, not accompanied by caspases-9 and -3 activation, probably due to 3BP-dependent alkylation of cysteine residues at caspase-9 catalytic site. To explore the possibility of sensitizing cells to 3BP treatment, we exploited 3BP effects on mitochondria by using 30 µM 3BP in association with antimycin A or menadione concentrations that in themselves exhibit poor toxicity. 3BP effect on cyt c release and cell vitality loss was potentiated due the greater oxidative stress induced by antimycin or menadione association with 3BP, supporting a preeminent role of mitochondrial ROS in 3BP toxicity. Indeed, the scavenger of mitochondrial superoxide MitoTEMPO counteracted 3BP-induced cyt c release and weakened the potentiating effect of 3BP/antimycin association. In conclusion, the biochemical mechanisms leading U118 glioblastoma cells to viability loss following 3BP treatment rely on mitochondrial ROS-dependent pathways. Their potentiation at low 3BP concentrations is consistent with the goal to minimize the toxic effect of the drug towards non-cancer cells.

Loss of cardiolipin in palmitate-treated GL15 glioblastoma cells favors cytochrome c release from mitochondria leading to apoptosis.

Unlike oleate and linoleate, palmitate induced mitochondrial apoptosis in GL15 glioblastoma cells. Decrease in membrane potential in a subpopulation of mitochondria of palmitate-treated cells was revealed using the 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide probe. The diminished ability to reduce a tetrazolium salt indicated an impairment of mitochondrial function. Up to 50% cytochrome c (cyt c) was detached from the inner mitochondrial membrane and released outside mitochondria in palmitate-treated cells, whereas no release was detected after oleate and linoleate treatments. Cyt c release into the cytosol was followed by caspase 3 activation. Released cyt c and caspase 3 activity were not affected by neutral and acid sphingomyelinase inhibitors and by the inhibitor of serine palmitoyltransferase cycloserine, indicating that apoptosis was independent of the ceramide pathway, nor the mitochondrial pro-apoptotic AIF or Bcl-2/Bax factors appeared to be involved in the effect. Utilization of palmitate by GL15 cells altered phospholipid composition. Cardiolipin (CL), the lipid involved in cyt c interaction with the inner mitochondrial membrane, was decreased and highly saturated. This produced an imbalance in hydrophilic/hydrophobic interactions underlying the anchorage of cyt c, by weakening the hydrophobic component and facilitating detachment of the protein and activation of downstream processes. The primary role of CL was explored by supplying GL15 with exogenous CL through a fusion process of CL liposomes with cell plasma membrane. Fused CL moved to mitochondria, as detected by nonylacridine orange probe. Enrichment of mitochondrial membranes with CL prior to palmitate treatment of cells caused decreased cyt c release and caspase 3 activity.

Molecular cloning and structural organization of the gene encoding the mouse lysosomal di-N-acetylchitobiase (ctbs).

The lysosomal enzyme di-N-acetylchitobiase hydrolyzes N-acetylglucosamine from the reducing-end of the N,N' diacetylchitobiose core of N-linked-oligosaccharides. The presence of chitobiase in the tissues of different species is probably responsible for differences in the structure of oligosaccharides accumulated in the lysosomal storage disease beta-mannosidosis. The disease has so far been described in humans, cats, cattle and goats. Low chitobiase activity has been observed in the tissues of ruminants and it has been hypothesized that in cattle this low level of expression is due to evolutionary changes in the promoter region. A cDNA encoding the mouse chitobiase has been isolated, sequenced and its identity confirmed by expression in COS-7 cells. Comparison of the mouse genomic sequence with the cDNA sequence revealed the presence of seven exons within the chitobiase gene. The gene spans about 15 kb and a single transcription initiation site was determined by 5'RACE. Chitobiase is differentially and ubiquitously expressed in mouse tissues as demonstrated by qRT-PCR analysis. Chitobiase is differentially expressed at lower levels in bovine tissues. In two bovine tissues (heart and muscle) mRNA was not detectable. Mouse and bovine promoters have been isolated and sequenced and their activities compared. The activity of the bovine promoter is very low and might explain the low activity of chitobiase observed in cattle.

Sterol dependent regulation of human TM7SF2 gene expression: role of the encoded 3beta-hydroxysterol Delta14-reductase in human cholesterol biosynthesis.

3Beta-hydroxysterol Delta(14)-reductase operates during the conversion of lanosterol to cholesterol in mammalian cells. Besides the endoplasmic reticulum 3beta-hydroxysterol Delta(14)-reductase (C14SR) encoded by TM7SF2 gene, the lamin B receptor (LBR) of the inner nuclear membrane possesses 3beta-hydroxysterol Delta(14)-reductase activity, based on its ability to complement C14SR-defective yeast strains. LBR was indicated as the primary 3beta-hydroxysterol Delta(14)-reductase in human cholesterol biosynthesis, since mutations in LBR gene were found in Greenberg skeletal dysplasia, characterized by accumulation of Delta(14)-unsaturated sterols. This study addresses the issue of C14SR and LBR role in cholesterol biosynthesis. Both human C14SR and LBR expressed in COS-1 cells exhibit 3beta-hydroxysterol Delta(14)-reductase activity in vitro. TM7SF2 mRNA and C14SR protein expression in HepG2 cells grown in delipidated serum (LPDS) plus lovastatin (sterol starvation) were 4- and 8-fold higher, respectively, than in LPDS plus 25-hydroxycholesterol (sterol feeding), resulting in 4-fold higher 3beta-hydroxysterol Delta(14)-reductase activity. No variations in LBR mRNA and protein levels were detected in the same conditions. The induction of TM7SF2 gene expression is turned-on by promoter activation in response to low cell sterol levels and is mediated by SREBP-2. The results suggest a primary role of C14SR in human cholesterol biosynthesis, whereas LBR role in the pathway remains unclear.

3-Bromopyruvate treatment induces alterations of metabolic and stress-related pathways in glioblastoma cells.

Glioblastoma (GBM) is the most common and aggressive brain tumour of adults. The metabolic phenotype of GBM cells is highly dependent on glycolysis; therefore, therapeutic strategies aimed at interfering with glycolytic pathways are under consideration. 3-Bromopyruvate (3BP) is a potent antiglycolytic agent, with a variety of targets and possible effects on global cell metabolism. Here we analyzed the changes in protein expression on a GBM cell line (GL15 cells) caused by 3BP treatment using a global proteomic approach. Validation of differential protein expression was performed with immunoblotting and enzyme activity assays in GL15 and U251 cell lines. The results show that treatment of GL15 cells with 3BP leads to extensive changes in the expression of glycolytic enzymes and stress related proteins. Importantly, other metabolisms were also affected, including pentose phosphate pathway, aminoacid synthesis, and glucose derivatives production. 3BP elicited the activation of stress response proteins, as shown by the phosphorylation of HSPB1 at serine 82, caused by the concomitant activation of the p38 pathway. Our results show that inhibition of glycolysis in GL15 cells by 3BP influences different but interconnected pathways. Proteome analysis may help in the molecular characterization of the glioblastoma response induced by pharmacological treatment with antiglycolytic agents. SIGNIFICANCE: Alteration of the glycolytic pathway characterizes glioblastoma (GBM), one of the most common brain tumours. Metabolic reprogramming with agents able to inhibit carbohydrate metabolism might be a viable strategy to complement the treatment of these tumours. The antiglycolytic agent 3-bromopyruvate (3BP) is able to strongly inhibit glycolysis but it may affect also other cellular pathways and its precise cellular targets are currently unknown. To understand the protein expression changes induced by 3BP, we performed a global proteomic analysis of a GBM cell line (GL15) treated with 3BP. We found that 3BP affected not only the glycolytic pathway, but also pathways sharing metabolic intermediates with glycolysis, such as the pentose phosphate pathway and aminoacid metabolism. Furthermore, changes in the expression of proteins linked to resistance to cell death and stress response were found. Our work is the first analysis on a global scale of the proteome changes induced by 3BP in a GBM model and may contribute to clarifying the anticancer potential of this drug.