Glucose-depleted Conditions Research Articles

Merlin/NF2 regulates SLC7A11/xCT expression and cell viability under glucose deprivation at high cell density in glioblastoma cells.

The cystine/glutamate transporter SLC7A11/xCT is highly expressed in many cancer cells and plays an important role in antioxidant activity by supplying cysteine for glutathione synthesis. Under glucose-depleted conditions, however, SLC7A11-mediated cystine uptake causes oxidative stress and cell death called disulfidptosis, a new form of cell death. We previously reported that high cell density (HD) promotes lysosomal degradation of SLC7A11 in glioblastoma cells, allowing them to survive under glucose-depleted conditions. In this study, we found that the neurofibromatosis type 2 gene, Merlin/NF2 is a key regulator of SLC7A11 in glioblastoma cells at HD. Deletion of Merlin increased SLC7A11 protein level and cystine uptake at HD, leading to promotion of cell death under glucose deprivation. Furthermore, HD significantly decreased SLC7A11 mRNA level, which was restored by Merlin deletion. This study suggests that Merlin suppresses glucose deprivation-induced cell death by downregulating SLC7A11 expression in glioblastoma cells at HD.

Mutation Status and Glucose Availability Affect the Response to Mitochondria-Targeted Quercetin Derivative in Breast Cancer Cells

Mitochondria, the main cellular power stations, are important modulators of redox-sensitive signaling pathways that may determine cell survival and cell death decisions. As mitochondrial function is essential for tumorigenesis and cancer progression, mitochondrial targeting has been proposed as an attractive anticancer strategy. In the present study, three mitochondria-targeted quercetin derivatives (mitQ3, 5, and 7) were synthesized and tested against six breast cancer cell lines with different mutation and receptor status, namely ER-positive MCF-7, HER2-positive SK-BR-3, and four triple-negative (TNBC) cells, i.e., MDA-MB-231, MDA-MB-468, BT-20, and Hs 578T cells. In general, the mito-quercetin response was modulated by the mutation status. In contrast to unmodified quercetin, 1 µM mitQ7 induced apoptosis in breast cancer cells. In MCF-7 cells, mitQ7-mediated apoptosis was potentiated under glucose-depleted conditions and was accompanied by elevated mitochondrial superoxide production, while AMPK activation-based energetic stress was associated with the alkalization of intracellular milieu and increased levels of NSUN4. Mito-quercetin also eliminated doxorubicin-induced senescent breast cancer cells, which was accompanied by the depolarization of mitochondrial transmembrane potential. Limited glucose availability also sensitized doxorubicin-induced senescent breast cancer cells to apoptosis. In conclusion, we show an increased cytotoxicity of mitochondria-targeted quercetin derivatives compared to unmodified quercetin against breast cancer cells with different mutation status that can be potentiated by modulating glucose availability.

GPRC5B (G protein-coupled receptor class C group 5 member B) suppresses glucose starvation-induced apoptosis in head-and-neck squamous cell carcinoma.

G protein-coupled receptor class C group 5 member B (GPRC5B) is involved in extracellular glucose sensing, glucose metabolism, and insulin resistance. Many cancers require glucose at high concentrations to survive and grow. We have investigated the association between tumour GPRC5B expression and the prognosis for patients with cancer, including head-and-neck squamous cell carcinoma (HNSCC), using data from The Human Protein Atlas. The 5-year survival rate was significantly reduced in patients with HNSCC, gastric, pancreatic, colorectal, and breast cancers if their tumours exhibited high levels of GPRC5B expression. The role of GPRC5B in glucose metabolism was assessed using six HNSCC cell lines with varying levels of GPRC5B expression. High levels of GPRC5B expression were found to favour rapid cell growth. The viability of an HNSCC cell line with normal and transfected GPRC5B expression was also assessed and no differences were observed under standard culture conditions. However, under glucose-deficient culture conditions, GPRC5B-overexpressing cells exhibited increased viability and reduced apoptosis. The results highlight the association between high GPRC5B expression and poor 5-year survival rates in patients with various cancers, including HNSCC. Furthermore, we have demonstrated that GPRC5B supports cancer cell survival under glucose-depleted conditions and could be a target molecule for cancer therapy.

Serum fatty acid-binding protein 5 is a significant factor in hepatocellular carcinoma progression independent of tissue expression level.

Fatty acid-binding protein 5 (FABP5) is highly expressed in hepatocellular carcinoma (HCC) tissues and is related to HCC progression. In this study, we analyzed the potential of serum FABP5 (sFABP5) as a tumor marker in HCC and its clinical significance in HCC progression. We compared the sFABP5 concentration in patients with HCC (HCC group) with that of patients with hepatitis without HCC (hepatitis group). Moreover, we measured the FABP5 expression levels in resected HCC tissues (tFABP5) and analyzed their relationship with sFABP5. We also performed cell-based assays using FABP5 knockout and overexpressing HCC cell lines to analyze the effect of extrinsic FABP5 (exFABP5) on HCC cells. We showed that sFABP5 was not a useful tumor marker for HCC, as HCC and sFABP5 were not correlated. However, sFABP5 and tFABP5 significantly correlated with survival after surgery for HCC, while sFABP5 and tFABP5 were independent of each other. In cell-based assays, exFABP5 was taken up by HCC cell lines and positively affected cell survival under glucose-depleted conditions by complementing the endogenous FABP5 function. In conclusion, sFABP5 had a significant impact on HCC progression irrespective of tFABP5 by augmenting cell viability under glucose-depleted conditions. As tFABP5 and sFABP5 are important factors that are independent of each other in HCC progression, both of them should be considered independently in improving the prognosis of patients with HCC.

Abstract B42: Identification of CPT1A as a novel driver of proliferation in luminal breast cancer

Abstract Clinically, approximately two-thirds of the nearly 250,000 breast cancer cases diagnosed each year in the United States are hormone receptor-positive, luminal breast tumors. Slower-growing luminal breast tumors are often successfully treated using endocrine-based therapies, resulting in a relatively good prognosis for these patients. However, more highly proliferative luminal tumors, even with the recent approval of CDK4/6 inhibitors, are often resistant, or become resistant, to current therapies, leading to a worse outcome for these women. Therefore, it is necessary to identify genetic events responsible for tumorigenesis and to identify potential druggable genetic alterations and/or pathways in order to improve clinical outcome. To address this question, we recently developed and used an innovative genomics-based strategy to interrogate orthogonal genome-wide data from more than 2,500 patients from the TCGA and METABRIC studies. By using an 11-gene mRNA-based gene expression signature of proliferation as a conceptual framework, we identified DNA copy number alterations and somatic mutations associated with luminal breast cancer proliferation. In order to identify the subset of amplified genes that are essential for cell viability, we analyzed a dataset of 27 breast cancer cell lines with available mRNA expression and genome-wide shRNA proliferation data. By integrating DNA copy number and shRNA analyses, we identified amplified carnitine palmitoyltransferase 1A (CPT1A) as a novel driver of proliferation in luminal breast cancer. CPT1A was found to be amplified in 40.2% of highly proliferative (top quartile) luminal tumors compared to 24.7% of all other samples and CPT1A DNA copy number status correlated with mRNA expression levels. Importantly, CPT1A was shown to be essential in luminal breast cancer cell lines with a high proliferation signature score. By analyzing RPPA data from luminal breast tumors, we confirmed CPT1A amplified tumors have increased expression of protein markers of proliferation. Since CPT1A is the rate-limiting enzyme responsible for fatty acid import into the mitochondria during fatty acid β oxidation (FAO), these data indicate that highly proliferative luminal tumors may utilize FAO as a prominent energy source. Interestingly, our analyses of RNA-seq data (n=1,031) demonstrated that highly proliferative luminal tumors have significantly higher CPT1A mRNA expression than either basal-like, which are largely synonymous with TNBC, or less proliferative luminal tumors, suggesting that these tumors may have a greater dependency on CPT1A activity. CPT1A has two major variants: cytoplasmic Variant 1 is the rate-limiting enzyme responsible for fatty acid import into the mitochondria during FAO while nuclear Variant 2 is purported to mediate histone deacetylase activity. Analyses of RNAseq data (n=819) demonstrated that Variant 1 represents >99.2% of CPT1A mRNA transcripts in a given tumor, suggesting the FAO-associated role of CPT1A is the more critical function of this protein in breast cancer. Consistent with this observation, we determined that breast cancer cell lines with high CPT1A protein expression are significantly more sensitive to etomoxir, a CPT1A-specific inhibitor known to repress FAO, when compared to cell lines with low CPT1A protein levels. Finally, we determined that under glucose-depleted conditions, cell lines with high CPT1A expression showed no difference in etomoxir response whereas cells with low CPT1A become significantly more sensitive. Collectively, these data suggest that CPT1A amplification and overexpression contribute to proliferation. Given that a number of approved or experimental drugs target CPT1A or FAO, our data suggest that CPT1A and/or FAO may represent a novel therapeutic target for luminal breast cancer. Citation Format: Kristina Mastrioanni, Kimberly Parker, Michael L. Gatza. Identification of CPT1A as a novel driver of proliferation in luminal breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B42.

Assessment of Anticancer Drug Effects on Pancreatic Cancer Cells under Glucose-Depleted Conditions Using Intracellular and Extracellular Amino Acid Metabolomics

Previously, we developed a method to evaluate states of cells treated with anticancer drugs via the comprehensive analysis of amino acids, termed amino acid metabolomics. In the present study, we evaluated the effects of the anticancer drugs, gemcitabine hydrochloride and pyrvinium pamoate, on the proliferation of a pancreatic cancer cell line (PANC-1) under hypoglycemic conditions using amino acid metabolomics. Intracellular and extracellular amino acid profiles of PANC-1 were determined by hydrophilic interaction chromatography-tandem mass spectrometry with simple pretreatment. Changes to the drugs' anticancer effects resulting from glucose starvation conditions were presented in score plots obtained from principal component analyses. In particular, the analysis of intracellular amino acids was found to be the superior approach because the results allowed a clearer assessment of the cell state. Further, orthogonal partial least squares discriminant analysis was performed to search for amino acid candidates that discriminate with anticancer drug-treated PANC-1 cells. We identified several amino acids that might be able to distinguish the drug-treated group from the control group. These results might provide a better understanding of the mechanisms underlying cell responses such as drug resistance or austerity. The present study is the first to evaluate the efficacy of anticancer drugs under glucose starvation based on the analysis of the variation of extracellular and intracellular amino acid profiles in vitro.

The stress-regulatory transcription factors Msn2 and Msn4 regulate fatty acid oxidation in budding yeast

The transcription factors Msn2 and Msn4 (multicopy suppressor of SNF1 mutation proteins 2 and 4) bind the stress-response element in gene promoters in the yeast Saccharomyces cerevisiae However, the roles of Msn2/4 in primary metabolic pathways such as fatty acid β-oxidation are unclear. Here, in silico analysis revealed that the promoters of most genes involved in the biogenesis, function, and regulation of the peroxisome contain Msn2/4-binding sites. We also found that transcript levels of MSN2/MSN4 are increased in glucose-depletion conditions and that during growth in nonpreferred carbon sources, Msn2 is constantly localized to the nucleus in wild-type cells. Of note, the double mutant msn2Δmsn4Δ exhibited a severe growth defect when grown with oleic acid as the sole carbon source and had reduced transcript levels of major β-oxidation genes. ChIP indicated that Msn2 has increased occupancy on the promoters of β-oxidation genes in glucose-depleted conditions, and in vivo reporter gene analysis indicated reduced expression of these genes in msn2Δmsn4Δ cells. Moreover, mobility shift assays revealed that Msn4 binds β-oxidation gene promoters. Immunofluorescence microscopy with anti-peroxisome membrane protein antibodies disclosed that the msn2Δmsn4Δ strain had fewer peroxisomes than the wild type, and lipid analysis indicated that the msn2Δmsn4Δ strain had increased triacylglycerol and steryl ester levels. Collectively, our data suggest that Msn2/Msn4 transcription factors activate expression of the genes involved in fatty acid oxidation. Because glucose sensing, signaling, and fatty acid β-oxidation pathways are evolutionarily conserved throughout eukaryotes, the msn2Δmsn4Δ strain could therefore be a good model system for further study of these critical processes.

Selective cytotoxicity of the anti-diabetic drug, metformin, in glucose-deprived chicken DT40 cells.

Metformin is a biguanide drug that is widely used in the treatment of diabetes. Epidemiological studies have indicated that metformin exhibits anti-cancer activity. However, the molecular mechanisms underlying this activity currently remain unclear. We hypothesized that metformin is cytotoxic in a tumor-specific environment such as glucose deprivation and/or low oxygen (O2) tension. We herein demonstrated that metformin was highly cytotoxic under glucose-depleted, but not hypoxic (2% O2) conditions. In order to elucidate the underlying mechanisms of this selective cytotoxicity, we treated exposed DNA repair-deficient chicken DT40 cells with metformin under glucose-depleted conditions and measured cellular sensitivity. Under glucose-depleted conditions, metformin specifically killed fancc and fancl cells that were deficient in FANCC and FANCL proteins, respectively, which are involved in DNA interstrand cross-link repair. An analysis of chromosomal aberrations in mitotic chromosome spreads revealed that a clinically relevant concentration of metformin induced DNA double-strand breaks (DSBs) in fancc and fancl cells under glucose-depleted conditions. In summary, metformin induced DNA damage under glucose-depleted conditions and selectively killed cells. This metformin-mediated selective toxicity may suppress the growth of malignant tumors that are intrinsically deprived of glucose.

Abstract P3-07-14: Targeting ERR-α regulated lactate metabolism eliminates drug-resistant breast cancer cells

Abstract Novel therapeutic strategies to eliminate chemo-resistant tumour cells responsible for the development of secondary lesions are essential in order to prevent breast cancer relapse. We have developed an in vitro model system enabling isolation of a putative metastasis-initiating breast cancer cell sub-population with a mitotically quiescent, drug-resistant phenotype. We hypothesise that this population is able to utilise oestrogen-related receptor-alpha (ERR-α)-regulated oxidative lactate metabolism and that inhibition of this critical survival pathway will result in elimination of tumour cells that have survived anti-cancer therapies. Flow cytofluormetric monitoring of Vybrant® DiD retention identified a mitotically quiescent sub-population (~0.05%) in MDA-MB-231 human breast cancer cells grown under high glucose (11mM) conditions. DiD-retaining cells accumulated in the G2/M phase of the cell cycle and were shown to be non-senescent following cytochemical analysis of β-galactosidase activity. Cytotoxicity assays with doxorubicin (0.40μM for 72 hours) demonstrated increased survival in the quiescent fraction (40.76%) compared with the rapidly dividing bulk cell population (1.63%, P ≤ 0.0001). Isolated drug-resistant cells contained a sub-fraction (~1%) that was able to form new clonal populations following cessation of treatment. Mitotically quiescent cells were cultured under high glucose (11mM) or glucose-depleted conditions with high lactate (22mM) in the presence of ERR-α inhibitors Cpd29 (10μM) or XCT790 (10μM). Colony formation was completely eliminated in Cpd29- and XCT790-treated samples compared to untreated controls. Drug-resistant sub-clones were isolated from the quiescent population following 72 hours treatment with doxorubicin (0.40μM) and were cultured under glucose-depleted conditions with high lactate (22mM) in the presence of ERR-α inhibitors Cpd29 (10μM) or XCT790 (10μM). In both instances, colony formation was completely prevented. We provide the first evidence that blocking cellular energy production through inhibition of ERR-α regulated oxidative lactate metabolism can eradicate a chemo-resistant, quiescent breast cancer cell population. Our data suggest that ERR-α inhibitors may be used in combination with chemotherapy to eliminate minimal residual disease and reduce breast cancer recurrence. Citation Format: Quayle L, Park S, McDonnell DP, Ottewell PD, Holen I. Targeting ERR-α regulated lactate metabolism eliminates drug-resistant breast cancer cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-14.

The role of nutrients in B lymphocyte growth and survival responses.

Abstract B cell activation is an energetically demanding process during which B lymphocytes shift from a resting state to a proliferative, metabolically active state. Little is known about the metabolic reprogramming process or the role extracellular nutrients play in the activation response. We demonstrate that there are distinct requirements for the nutrients L-glutamine and glucose during activation. We show that cells activated in glucose-depleted conditions are still able to undergo growth and signaling events. In contrast, we show that extracellular L-glutamine is essential for all but the earliest activation events, and cells cultured in L-glutamine-deprived conditions are unable to enter the cell cycle. Consistently, we show that extracellular supplementation of the cell-permeable derivative of a-ketoglutarate (a-KG) is able to rescue cell activation in the absence of glutamine. We also show the induction of the high affinity amino acid transporter ASCT2 is required for glutamine uptake following BCR crosslinking. Specifically, we found that halting glutamine uptake or processing by inhibiting ASCT2 or the glutaminolytic enzyme glutaminase causes activation defects that parallel those observed in glutamine deprived conditions, indicating a requirement for glutaminolysis during the very early stages of activation. Finally, we found that a-KG does not contribute to epigenetic remodeling, but is necessary for mTORC1 activation. In turn, mTORC1 activity is required for upregulation of the glucose transporter Glut1 during activation, as well as increased glucose uptake. These findings indicate a distinct metabolic profile that begins with glutamine uptake, and acts through mTORC1 signaling to later promote glucose uptake.

PTEN regulates plasma membrane expression of glucose transporter 1 and glucose uptake in thyroid cancer cells.

Glucose represents an important source of energy for the cells. Proliferating cancer cells consume elevated quantity of glucose, which is converted into lactate regardless of the presence of oxygen. This phenomenon, known as the Warburg effect, has been proven to be useful for imaging metabolically active tumours in cancer patients by (18)F-fluorodeoxyglucose positron emission tomography (FDG-PET). Glucose is internalised in the cells by glucose transporters (GLUTs) belonging to the GLUT family. GLUT1 (SLC2A1) is the most prevalent isoform in more aggressive and less differentiated thyroid cancer histotypes. In a previous work, we found that loss of expression of PTEN was associated with increased expression of GLUT1 on the plasma membrane (PM) and probability of detecting thyroid incidentalomas by FDG-PET. Herein, we investigated the molecular pathways that govern the expression of GLUT1 on the PM and the glucose uptake in WRO (expressing WT PTEN) and FTC133 (PTEN null) follicular thyroid cancer cells cultured under glucose-depleted conditions. The membrane expression of GLUT1 was enhanced in glucose-deprived cells. Through genetic manipulations of PTEN expression, we could demonstrate that the lack of this oncosuppressor has a dominant effect on the membrane expression of GLUT1 and glucose uptake. We conclude that loss of function of PTEN increases the probability of cancer detection by FDG-PET or other glucose-based imaging diagnosis.

Inhibitors of the glyoxylate cycle enzyme ICL1 in Candida albicans for potential use as antifungal agents.

Candida albicans is an opportunistic pathogen that causes candidiasis in humans. In recent years, metabolic pathways in C. albicans have been explored as potential antifungal targets to treat candidiasis. The glyoxylate cycle, which enables C. albicans to survive in nutrient-limited host niches and its. Key enzymes (e.g., isocitrate lyase (ICL1), are particularly attractive antifungal targets for C. albicans. In this study, we used a new screening approach that better reflects the physiological environment that C. albicans cells experience during infection to identify potential inhibitors of ICL. Three compounds (caffeic acid (CAFF), rosmarinic acid (ROS), and apigenin (API)) were found to have antifungal activity against C. albicans when tested under glucose-depleted conditions. We further confirmed the inhibitory potential of these compounds against ICL using the ICL enzyme assay. Lastly, we assessed the bioavailability and toxicity of these compounds using Lipinski's rule-of-five and ADMET analysis.

Cytosolic NADPH Homeostasis in Glucose-starved Procyclic Trypanosoma brucei Relies on Malic Enzyme and the Pentose Phosphate Pathway Fed by Gluconeogenic Flux

All living organisms depend on NADPH production to feed essential biosyntheses and for oxidative stress defense. Protozoan parasites such as the sleeping sickness pathogen Trypanosoma brucei adapt to different host environments, carbon sources, and oxidative stresses during their infectious life cycle. The procyclic stage develops in the midgut of the tsetse insect vector, where they rely on proline as carbon source, although they prefer glucose when grown in rich media. Here, we investigate the flexible and carbon source-dependent use of NADPH synthesis pathways in the cytosol of the procyclic stage. The T. brucei genome encodes two cytosolic NADPH-producing pathways, the pentose phosphate pathway (PPP) and the NADP-dependent malic enzyme (MEc). Reverse genetic blocking of those pathways and a specific inhibitor (dehydroepiandrosterone) of glucose-6-phosphate dehydrogenase together established redundancy with respect to H2O2 stress management and parasite growth. Blocking both pathways resulted in ∼10-fold increase of susceptibility to H2O2 stress and cell death. Unexpectedly, the same pathway redundancy was observed in glucose-rich and glucose-depleted conditions, suggesting that gluconeogenesis can feed the PPP to provide NADPH. This was confirmed by (i) a lethal phenotype of RNAi-mediated depletion of glucose-6-phosphate isomerase (PGI) in the glucose-depleted Δmec/Δmec null background, (ii) an ∼10-fold increase of susceptibility to H2O2 stress observed for the Δmec/Δmec/(RNAi)PGI double mutant when compared with the single mutants, and (iii) the (13)C enrichment of glycolytic and PPP intermediates from cells incubated with [U-(13)C]proline, in the absence of glucose. Gluconeogenesis-supported NADPH supply may also be important for nucleotide and glycoconjugate syntheses in the insect host.

Glucose-induced Remodeling of Intermediary and Energy Metabolism in Procyclic Trypanosoma brucei

The procyclic form of Trypanosoma brucei is a parasitic protozoan that normally dwells in the midgut of its insect vector. In vitro, this parasite prefers d-glucose to l -proline as a carbon source, although this amino acid is the main carbon source available in its natural habitat. Here, we investigated how l -proline is metabolized in glucose-rich and glucose-depleted conditions. Analysis of the excreted end products of (13)C-enriched l -proline metabolism showed that the amino acid is converted into succinate or l -alanine depending on the presence or absence of d-glucose, respectively. The fact that the pathway of l -proline metabolism was truncated in glucose-rich conditions was confirmed by the analysis of 13 separate RNA interference-harboring or knock-out cell lines affecting different steps of this pathway. For instance, RNA interference studies revealed the loss of succinate dehydrogenase activity to be conditionally lethal only in the absence of d-glucose, confirming that in glucose-depleted conditions, l -proline needs to be converted beyond succinate. In addition, depletion of the F(0)/F(1)-ATP synthase activity by RNA interference led to cell death in glucose-depleted medium, but not in glucose-rich medium. This implies that, in the presence of d-glucose, the importance of the F(0)/F(1)-ATP synthase is diminished and ATP is produced by substrate level phosphorylation. We conclude that trypanosomes develop an elaborate adaptation of their energy production pathways in response to carbon source availability.

Regulating Snf1 Localization

Snf1, a serine-threonine kinase in Saccharomyces cerevisiae, is activated under conditions of glucose deprivation and regulates the expression of specific genes involved in cellular metabolism. Snf1 can control the activity of specific transcriptional activators and repressors and of RNA polymerase II in the nucleus. Snf1 protein kinases exist as heterotrimers with a γ subunit and one of three different β subunits--Gal83, Sip1, or Sip2--but the significance of each interaction is unclear. Vincent et al. showed that under glucose-depleted conditions, expressed chimeric proteins consisting of green fluorescent protein (GFP) fused to Sip1 or Sip2 localized to the vacuole or cytoplasm, respectively, whereas GFP-Gal83 localized to the nucleus, suggesting that Gal83 targets Snf1 to the nucleus. GFP-Snf1 also localized to the nucleus in glucose-depleted conditions. Translocation of Snf1-Gal83 to the nucleus was independent of the kinase activity of Snf1. In the presence of glucose, Snf1-Gal83 was excluded from the nucleus in a manner dependent on glucose phosphorylation. The authors note that independent regulation of the activity and localization of Snf1 should enhance the versatility of physiological regulation mediated by this protein kinase.O. Vincent, R. Townley, S. Kuchin, M. Carlson, Subcellular localization of the Snf1 kinase is regulated by specific β subunits and a novel glucose signaling mechanism. Genes Dev. 15, 1104-1114 (2001). [Abstract] [Full Text]

Regulating Snf1 Localization

Snf1, a serine-threonine kinase in Saccharomyces cerevisiae , is activated under conditions of glucose deprivation and regulates the expression of specific genes involved in cellular metabolism. Snf1 can control the activity of specific transcriptional activators and repressors and of RNA polymerase II in the nucleus. Snf1 protein kinases exist as heterotrimers with a γ subunit and one of three different β subunits--Gal83, Sip1, or Sip2--but the significance of each interaction is unclear. Vincent et al. showed that under glucose-depleted conditions, expressed chimeric proteins consisting of green fluorescent protein (GFP) fused to Sip1 or Sip2 localized to the vacuole or cytoplasm, respectively, whereas GFP-Gal83 localized to the nucleus, suggesting that Gal83 targets Snf1 to the nucleus. GFP-Snf1 also localized to the nucleus in glucose-depleted conditions. Translocation of Snf1-Gal83 to the nucleus was independent of the kinase activity of Snf1. In the presence of glucose, Snf1-Gal83 was excluded from the nucleus in a manner dependent on glucose phosphorylation. The authors note that independent regulation of the activity and localization of Snf1 should enhance the versatility of physiological regulation mediated by this protein kinase. O. Vincent, R. Townley, S. Kuchin, M. Carlson, Subcellular localization of the Snf1 kinase is regulated by specific β subunits and a novel glucose signaling mechanism. Genes Dev. 15 , 1104-1114 (2001). [Abstract] [Full Text]

Improved gene expression inAspergillus nidulans

A variety of gene expression systems have been developed that utilize the promoter and transcriptional regulatory sequences derived from carbon-catabolite repressed genes for the expression of heterologous genes. The alcA expression system of Aspergillus nidulans utilizes the promoter and regulatory sequences derived from the alcohol dehydrogenase I (alcA) gene. Expression of the alcA gene is repressed by a DNA-binding protein (CreA) in the presence of glucose and induced by ethanol under glucose-depleted conditions. One problem encountered during the expression of therapeutic proteins in A. nidulans is the coexpression of secreted proteases at the time of maximal secretion of heterologous product. To avoid the proteases we created an alcA promoter variant that is no longer sensitive to glucose repression hence could drive expression at earlier time points during the fermentation. The use of this promoter variant in the expression of recombinant interleukin-6 is discussed. A second problem encountered during the expression of high-quality human therapeutic proteins in Aspergillus is aberrant glycosylation. Lower eukaryotic systems, such as Aspergillus, tend to add highly branched mannosidic chains to heterologous secreted protein products. N-Glycans can be important for both the structure and function of specific glycoproteins, hence efforts are being made to in vivo alter the type and complexity of N-glycans substituted by A. nidulans. Key words: Aspergillus, gene expression, alcohol dehydrogenase, glycosylation.