L-2hydroxyglutaric acid rewires amino acid metabolism in colorectal cancer via the mTOR-ATF4 axis.

Oncometabolites, such as D/L-2-hydroxyglutarate (2HG), have directly been implicated in carcinogenesis; however, the underlying molecular mechanisms remain poorly understood. Here, we showed that the levels of the L-enantiomer of 2HG (L2HG) were specifically increased in colorectal cancer (CRC) tissues and cell lines compared with the D-enantiomer of 2HG (D2HG). In addition, L2HG increased the expression of ATF4 and its target genes by activating the mTOR pathway, which subsequently provided amino acids and improved the survival of CRC cells under serum deprivation. Downregulating the expression of L-2-hydroxyglutarate dehydrogenase (L2HGDH) and oxoglutarate dehydrogenase (OGDH) increased L2HG levels in CRC, thereby activating mTOR-ATF4 signaling. Furthermore, L2HGDH overexpression reduced L2HG-mediated mTOR-ATF4 signaling under hypoxia, whereas L2HGDH knockdown promoted tumor growth and amino acid metabolism in vivo. Together, these results indicate that L2HG ameliorates nutritional stress by activating the mTOR-ATF4 axis and thus could be a potential therapeutic target for CRC.

Direct protein-protein interaction between Npas4 and IPAS mutually inhibits their critical roles in neuronal cell survival and death.

Inhibitory PAS domain protein (IPAS) is a bifunctional protein that acts as a transcriptional repressor in hypoxia and as a pro-apoptotic protein involved in neuronal cell death. Npas4 (NXF or LE-PAS) is a transcriptional factor that protects nerve cells from endogenous and foreign neurotoxins. Here we show that IPAS and Npas4 antagonize each other through their direct interaction. Coimmunoprecipitation experiments revealed that multiple binding sites on each protein were involved in the interaction. CoCl2 treatment of PC12 cells that induces IPAS repressed the transactivation activity of Npas4, and IPAS siRNA treatment reduced the CoCl2-induced repression. CoCl2-induced apoptosis was suppressed by the addition of KCl that induces Npas4. The protective effect of KCl was attenuated by siRNA-mediated gene silencing of Npas4. Npas4 and IPAS proteins were induced and localized in the cytoplasm of the dopaminergic neurons in the substantia nigra pars compacta after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment. Npas4-/- mice exhibited greater sensitivity to MPTP in nigral dopaminergic neurons. Together, these results strongly suggest that neuroprotective activity of Npas4 was, at least partly, exerted by inhibiting the pro-apoptotic activity of IPAS through direct interaction.

Attenuation of inhibitory PAS domain protein-induced cell death by synthetic peptides derived from Mcl-1 transmenbrane domain.

Expression of Inhibitory PAS domain protein (IPAS) induces apoptosis by inhibiting the anti-apoptotic activity of mitochondrial pro-survival proteins including Bcl-xL and Mcl-1 through direct binding. Analysis to examine the IPAS-binding region in Bcl-xL demonstrated that the C-terminal transmembrane (TM) domain is indispensable for the specific binding. A chimeric protein composed of the TM domain of Mcl-1 fused to the C-terminus of Citrine also exhibited a binding affinity to IPAS, and markedly attenuated apoptosis caused by the overexpression of Cerulean-IPAS in SH-SY5Y cells. HIV-1 TAT cell-penetrating peptide-conjugated synthetic peptides that cover whole or parts of the Mcl-1 TM domain showed anti-apoptotic activity in the CoCl2-induced cell death in PC12 cells. Administration of these highly effective anti-apoptotic peptides to mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that produces a reliable mouse model of Parkinson's disease (PD) decreased neuronal cell loss in the substantia nigra pars compacta. Therefore, the peptides may be considered promising therapeutic agents for neurodegenerative disorders such as PD and stroke.

Mitochondrial E3 Ubiquitin Ligase Parkin: Relationships with Other Causal Proteins in Familial Parkinson's Disease and Its Substrate-Involved Mouse Experimental Models.

Parkinson's disease (PD) is a common neurodegenerative disorder. Recent identification of genes linked to familial forms of PD has revealed that post-translational modifications, such as phosphorylation and ubiquitination of proteins, are key factors in disease pathogenesis. In PD, E3 ubiquitin ligase Parkin and the serine/threonine-protein kinase PTEN-induced kinase 1 (PINK1) mediate the mitophagy pathway for mitochondrial quality control via phosphorylation and ubiquitination of their substrates. In this review, we first focus on well-characterized PINK1 phosphorylation motifs. Second, we describe our findings concerning relationships between Parkin and HtrA2/Omi, a protein involved in familial PD. Third, we describe our findings regarding inhibitory PAS (Per/Arnt/Sim) domain protein (IPAS), a member of PINK1 and Parkin substrates, involved in neurodegeneration during PD. IPAS is a dual-function protein involved in transcriptional repression of hypoxic responses and the pro-apoptotic activities.

Increase in proapoptotic activity of inhibitory PAS domain protein via phosphorylation by MK2.

Inhibitory PAS domain protein (IPAS) is a bifunctional protein that downregulates hypoxic gene expression and exerts proapoptotic activity by preventing prosurvival activity of Bcl-xL and its related factors. Proapoptotic activity of IPAS is attenuated by the activation of the PINK1-Parkin pathway, and involved in neuronal degeneration in an experimental mouse model of Parkinson's disease. The current study shows that phosphorylation of IPAS at Ser184 by MAPK-activated protein kinase 2 (MK2 or MAPKAPK2) enhances the proapoptotic function of IPAS. Perinuclear clustering of mitochondria and activation of caspase-3 caused by the transient expression of EGFP-IPAS were increased by UVB irradiation. The C-terminal region of IPAS mediated the UVB susceptibility of IPAS. Increase in IPAS-induced mitochondrial clustering by UVB was completly inhibited by the p38 MAPK inhibitor SB203580. Mass spectrometry analysis of UVB-activated IPAS identified several phosphorylation sites in the C-terminal region containing p38 MAPK consensus phosphorylation sites at Ser219 and Ser223, and an MK2 consensus site at Ser184. Although mutations of Ser219 and Ser223 to Ala did not suppress the UVB-induced mitochondrial clustering, replacement of Ser184 with Ala blocked it. A phosphomimetic substitution at Ser184 enhanced mitochondrial clustering and activation of caspase-3 without UVB exposure. Furthermore, binding affinity to Bcl-xL was increased by the mutation. Treatment of PC12 cells with CoCl2 caused activation of MK2 and mitochondrial clustering. IPAS-dependent cell death induced by CoCl2 in PC12 cells was decreased by the treatment with the MK2 inhibitor MK2 inhibitor III and by siRNA-directed silencing of MK2.

Conformational changes in inhibitory PAS domain protein associated with binding of HIF-1α and Bcl-xL in living cells.

Inhibitory PAS domain protein (IPAS) is a dual function protein acting as a transcriptional repressor and as a pro-apoptotic protein. Simultaneous dual-color single-molecule imaging of EGFP-IPAS coexpressed with Mit-TagRFP-T in living HeLa cells revealed that fraction of EGFP-IPAS was arrested in the nucleus and on mitochondria. Transiently expressed Cerulean-IPAS in HEK293T cells was present in nuclear speckles when coexpressed with Citrine-HIF-1α or Citrine-HLF. Fluorescence lifetime imaging microscopy (FLIM) analysis of Citrine-IPAS-Cerulean in living CHO-K1 cells clarified the presence of intramolecular FRET. Reduced lifetimes of the donor were partially restored by coexpression of HIF-1α or Bcl-xL, binding proteins of IPAS in the nucleus and mitochondria, respectively. This alteration in lifetimes demonstrates that conformational changes occurred in IPAS by their binding.

Chimeric RNA Oligonucleotides Incorporating Triazole-Linked Trinucleotides: Synthesis and Function as mRNA in Cell-Free Translation Reactions.

A method for the synthesis of chimeric oligonucleotides was developed to incorporate purine nucleobases and multiple triazole linkers in natural, phosphate-linked structures of RNA. A solution-phase synthesis method for triazole-linked RNA oligomers via copper-catalyzed azide-alkyne cycloaddition reaction was optimized and tolerated purine nucleobases and protecting groups for further transformations. Three TLRNA trinucleotides with 5'-protected hydroxy and 3'-phosphoramidite groups were prepared, and one congener with a representative sequence was subjected to automated, solid-phase phosphoramidite synthesis. The synthesis allowed the efficient preparation of 13-mer chimeric RNA oligonucleotides with two triazole linkers, ten phosphate linkers and purine/pyrimidine nucleobases. The chimeric oligonucleotide was found applicable to a cell-free translation system as mRNA and provided the genetic code for dipeptide production.

Nucleocytoplasmic shuttling of IPAS by its unique nuclear import and export signals unshared with other HIF-3α splice variants.

Inhibitory Per/Arnt/Sim (PAS) domain protein (IPAS) is a splice variant of hypoxia-inducible factor (HIF)-3α, and possesses two entirely different functions. One is as a transcriptional repressor against HIF-dependent hypoxic gene activation. The other is as a pro-apoptotic factor by direct binding to the pro-survival protein Bcl-xL and its related proteins on mitochondria. Presently, the regulatory mechanism that determines the intracellular distribution of IPAS to fulfill each of the two functions is unknown. As a first step towards elucidation of the mechanism, nucleocytoplasmic transport signals of IPAS were explored. A bipartite-like nuclear localization signal (NLS) was found in the N-terminal region by the deletion and mutation analysis of EGFP-IPAS. In addition, the helix-loop-helix domain showed weak nuclear import/retention activity. A leptomycin B-sensitive nuclear export signal (NES) was localized in the C-terminal region of the protein. A proline-rich region supported the NES activity. These NLS and NES are not carried by the other variants of HIF-3α due to differential exon usage. These results strongly suggest that IPAS is a nucleocytoplasmic shuttling protein.

PHD1 interacts with ATF4 and negatively regulates its transcriptional activity without prolyl hydroxylation.

Cellular response to hypoxia plays an important role in both circulatory and pulmonary diseases and cancer. Hypoxia-inducible factors (HIFs) are major transcription factors regulating the response to hypoxia. The α-subunits of HIFs are hydroxylated by members of the prolyl-4-hydroxylase domain (PHD) family, PHD1, PHD2, and PHD3, in an oxygen-dependent manner. Here, we report on the identification of ATF4 as a protein interacting with PHD1 as well as PHD3, but not with PHD2. The central region of ATF4 including the Zipper II domain, ODD domain and ß-TrCP recognition motif were involved in the interaction with PHD1. Coexistence of PHD1 stabilized ATF4, as opposed to the destabilization of ATF4 by PHD3. Moreover, coexpression of ATF4 destabilized PHD3, whereas PHD1 stability was not affected by the presence of ATF4. Mutations to alanine of proline residues in ATF4 that satisfied hydroxylation consensus by PHDs did not affect binding activity of ATF4 to PHD1 and PHD3. Furthermore, in vitro prolyl hydroxylation assay clearly indicated that ATF4 did not serve as a substrate of both PHD1 and PHD3. Coexpression of PHD1 or PHD3 with ATF4 repressed the transcriptional activity of ATF4. These results suggest that PHD1 and PHD3 control the transactivation activity of ATF4.

Triazole-linked DNA as a primer surrogate in the synthesis of first-strand cDNA.

A phosphate-eliminated nonnatural oligonucleotide serves as a primer surrogate in reverse transcription reaction of mRNA. Despite of the nonnatural triazole linkages in the surrogate, the reverse transcriptase effectively elongated cDNA sequences on the 3'-downstream of the primer by transcription of the complementary sequence of mRNA. A structure-activity comparison with the reference natural oligonucleotides shows the superior priming activity of the surrogate containing triazole-linkages. The nonnatural linkages also protect the transcribed cDNA from digestion reactions with 5'-exonuclease and enable us to remove noise transcripts of unknown origins.

Tumour necrosis factor-α suppresses the hypoxic response by NF-κB-dependent induction of inhibitory PAS domain protein in PC12 cells.

Inflammation is often accompanied by hypoxia. However, crosstalk between signalling pathways activated by inflammation and signalling events that control adaptive response to hypoxia is not fully understood. Here we show that exposure to tumour necrosis factor-α (TNF-α) activates expression of the inhibitory PAS domain protein (IPAS) to suppress the hypoxic response caused by hypoxia-inducible factor (HIF)-1 and HIF-2 in rat pheochromocytoma PC12 cells but not in human hepatoma Hep3B cells. This induction of IPAS was dependent on the nuclear factor-κB (NF-κB) pathway and attenuated hypoxic induction of HIF-1 target genes such as tyrosine hydroxylase (TH) and vascular endothelial growth factor (VEGF). HIF-dependent reporter activity in hypoxia was also decreased following TNF-α treatment. Knockdown of IPAS mRNA by small interfering RNA (siRNA) restored the TNF-α-suppressed hypoxic response. These results indicate that TNF-α is a cell-type specific suppressor of HIFs and suggest a novel crosstalk between stimulation by inflammatory mediators and HIF-dependent hypoxic response.

Inhibitory effect of extracellular histidine on cobalt-induced HIF-1alpha expression.

Cobalt chloride (CoCl(2)) can mimic hypoxia in inducing hypoxia-inducible factor 1 (HIF-1). Several cultured cells were examined for susceptibility to CoCl(2) in DMEM, MEM and RPMI 1640 medium. Here we report that HIF-1α expression of mammalian cells by CoCl(2) was largely dependent on the culture medium. HIF-1α protein and hypoxia response element (HRE)-dependent reporter activity were strongly induced in RPMI 1640 but not in DMEM in several cultured cells including MCF-7, a human breast cancer cell line. Analysis of causal nutrients has revealed that histidine, which is contained richer in DMEM, acts as the inhibitory nutrient for cobalt-induced HIF-1α expression of MCF-7 cells in DMEM. D-Histidine also inhibited the HIF-1α activity at the same level as L-histidine, suggesting that sequestration of free cobaltous ion by chelation with histidine was the cause of the inhibition. These results demonstrate that selection of the culture medium must be considered with caution in cell culture experiments using CoCl(2) as a hypoxia-mimetic reagent.

Loose interaction between glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase revealed by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy in living cells.

Loose interaction between the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK) was visualized in living CHO-K1 cells by fluorescence resonance energy transfer (FRET), using time-domain fluorescence lifetime imaging microscopy. FRET between active tetrameric subunits of GAPDH linked to cerulean or citrine was observed, and this FRET signal was significantly attenuated by coexpression of PGK. Also, direct interaction between GAPDH-citrine and PGK-cerulean was observed by FRET. The strength of FRET signals between them was dependent on linkers that connect GAPDH to citrine and PGK to cerulean. A coimmunoprecipitation assay using hemagglutinin-tagged GAPDH and FLAG-tagged PGK coexpressed in CHO-K1 cells supported the FRET observation. Taken together, these results demonstrate that a complex of GAPDH and PGK is formed in the cytoplasm of living cells.

Polymorphisms and functional differences in aryl hydrocarbon receptors (AhR) in Japanese field mice, Apodemus speciosus.

Dioxins, which are unintentionally generated toxic pollutants, exert a variety of adverse effects on organisms. The majority of these effects, which include teratogenesis, immunosuppression, tumor promotion, and endocrine disruption, are mediated through aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor. Genetic variations in AhR result in different survivability under exposure to dioxin contamination, which might affect the genetic structure of wildlife populations through differential susceptibility to dioxin exposure. The aim of this study was to clarify the polymorphisms of AhR in Japanese field mice, Apodemus speciosus, and their functional differences in order to develop a molecular indicator for dioxin sensitivity. Wild Japanese field mice had abundant polymorphisms in AhR coding region. Seventy-one single nucleotide polymorphisms, 27 of which occur amino acid substitutions, and consequently 49 alleles were identified in 63 individuals. In the functional analysis of AhR variants using transient reporter assays, a Gln to Arg mutation at amino acid 799 exhibited a significant decrease in the level of transactivational properties (p=0.015) which might modify the dioxin susceptibility of an individual.

Transactivation activity of LBP-1 proteins and their dimerization in living cells.

LBP-1 proteins form dimers and act as transcription factors that activate a number of genes related to cell growth and differentiation. LBP-1a and LBP-1c are localized in the cytoplasm when transiently expressed in cultured cells, but translocated into the nucleus after forming heterodimers with LBP-1b, which is a splicing variant of LBP-1a with an intrinsic nuclear localization signal (NLS). Here, we report that LBP-1b showed potent transactivation activity, and that forcibly expressed LBP-1a and LBP-1c in the nucleus essentially exhibited very little or no transactivation activity. Mutations in the NLS that abolished the NLS activity of LBP-1b also abrogated the transactivation activity. We have found that LBP-1 proteins contain a putative sterile alpha motif domain indispensable for their dimerization capability in the C-terminal region. To demonstrate whether homo- and heterodimers composed of LBP-1a and/or LBP-1c are generated in the nucleus, we applied the FLIM-based fluorescence resonance energy transfer imaging technique to living cells. It revealed that dimers composed of LBP-1a and LBP-1c were re-formed probably by a partner-exchange of LBP-1b-containing heterodimers.

Cyclic AMP represses the hypoxic induction of hypoxia-inducible factors in PC12 cells.

Hypoxia-inducible factor 1 (HIF-1) is a master regulator for hypoxic activation of genes for angiogenesis, hormone synthesis, glycolysis and cell survival. In addition to hypoxic stimulus, various effectors and reagents were reported to affect HIF-1 activity. Here, we show that cyclic AMP (cAMP) down-regulates the HIF-1 activity in pheochromocytoma PC12 cells but not in Hep3B and HeLa cells. Hypoxia response element-dependent reporter activity was decreased by the addition of dibutyryl cAMP. Expression of protein kinase A (PKA) catalytic alpha-subunits repressed the HIF-1 activity. HIF-1alpha and HLF (HIF-2alpha or EPAS1) protein levels were decreased by the treatment with dibutyryl cAMP. Although CREB was served as a negative factor for the HIF-1 activity, it may not be a major PKA target in the cAMP-dependent HIF-alpha repression pathway. Induction of hypoxia responsive genes was suppressed by dibutyryl cAMP. Our results provide additional insight into a regulatory mechanism of hypoxic response.

Magnesium deficiency causes loss of response to intermittent hypoxia in paraganglion cells.

Magnesium deficiency is suggested to contribute to many age-related diseases. Hypoxia-inducible factor 1alpha (HIF-1alpha) is known to be a master regulator of hypoxic response. Here we show that hypomagnesemia suppresses reactive oxygen species (ROS)-induced HIF-1alpha activity in paraganglion cells of the adrenal medulla and carotid body. In PC12 cells cultured in the low magnesium medium and treated with cobalt chloride (CoCl(2)) or exposed to intermittent hypoxia, ROS-mediated HIF-1alpha activity was suppressed. This suppression was due to up-regulation of inhibitory PAS (Per/Arnt/Sim) domain protein (IPAS) that was caused by NF-kappaB activation, which resulted from ROS and calcium influx mainly through the T-type calcium channels. Induction of tyrosine hydroxylase, a target of HIF-1, by CoCl(2) injection was suppressed in the adrenal medulla of magnesium-deficient mice because of up-regulation of IPAS. Also in the carotid body of magnesium-deficient mice, CoCl(2) and chronic intermittent hypoxia failed to enhance the tyrosine hydroxylase expression. These results demonstrate that serum magnesium levels are a key determinant for ROS-induced hypoxic responses.

Role of the intracellular localization of HIF-prolyl hydroxylases.

Hypoxia-inducible factor-1 (HIF-1) is a major transcription factor regulating the response of tumor cells to hypoxia and is comprised of HIF-1alpha and Arnt (HIF-1beta). In mammalian cells, HIF-1 protein levels are regulated by three HIF-prolyl hydroxylases, termed PHD1, PHD2 and PHD3. To assess whether intracellular localization of PHD1 and PHD2 affects the hypoxic response via HIF-1, we investigated the localization signal of PHDs. PHD1 possessed at least one nuclear localization signal (NLS), and PHD2 contained a region as essential for nuclear export in their N-terminal region. Treatment of cells with leptomycin B revealed that PHD2 was able to shuttle between the cytoplasm and the nucleus. Reporter assay indicated that differences in the intracellular distribution of PHD1 did not influence on HIF-1alpha activity. However, a PHD2 mutant lacking the region for nuclear export exhibited significantly reduced effect to HIF-1alpha activity compared to wild-type PHD2, suggesting that the regulation of the intracellular distribution of PHD2 is an effective pathway for the control of the hypoxic response.

Expression, purification and characterization of human PHD1 in Escherichia coli.

The hypoxia-inducible factors (HIFs) play a central role in oxygen homeostasis. HIF prolyl hydroxylases (PHDs) modify HIFalpha subunits and thereby target them for proteasomal degradation. Mammalian PHDs comprise three isozymes, PHD1, PHD2 and PHD3, and belong to the iron(II)-2-oxoglutarate-dependent dioxygenase family. We have expressed full-length human PHD1 in Escherichia coli, and purified it to apparent homogeneity by immobilized Ni-affinity chromatography, cation-exchange HPLC followed by gel filtration. Fe(2+) was found to have EC(50) value of 0.64 microM and the purified enzyme showed maximal activity at 10 microM Fe(2+). The IC(50) values for transition metal ions, Co(2+), Ni(2+) and Cu(2+), were 58, 35 and 220 microM, respectively, in the presence of 100 microM Fe(2+). Mn(2+) did not affect the activity <1 mM. Many transcription-related proteins are regulated by phosphorylation. Thus, recombinant PHD1 was examined for in vitro phosphorylation using protein kinase A, protein kinase Calpha, casein kinase I and II and Erk2. The protein was most strongly phosphorylated by protein kinase Calpha, and the phosphorylation sites were found to be Ser-132, Ser-226 and Ser-234. Mutation of Ser-132 or Ser-234 to Asp or Glu diminished the enzymatic activity to 25-60%, while mutation of Ser-226 scarcely influenced the activity.

Dickkopf 1 (DKK1) regulates skin pigmentation and thickness by affecting Wnt/beta-catenin signaling in keratinocytes.

The epidermis (containing primarily keratinocytes and melanocytes) overlies the dermis (containing primarily fibroblasts) of human skin. We previously reported that dickkopf 1 (DKK1) secreted by fibroblasts in the dermis elicits the hypopigmented phenotype of palmoplantar skin due to suppression of melanocyte function and growth via the regulation of two important signaling factors, microphthalmia-associated transcription factor (MITF) and beta-catenin. We now report that treatment of keratinocytes with DKK1 increases their proliferation and decreases their uptake of melanin and that treatment of reconstructed skin with DKK1 induces a thicker and less pigmented epidermis. DNA microarray analysis revealed many genes regulated by DKK1, and several with critical expression patterns were validated by reverse transcriptase-polymerase chain reaction and Western blotting. DKK1 induced the expression of keratin 9 and alpha-Kelch-like ECT2 interacting protein (alphaKLEIP) but down-regulated the expression of beta-catenin, glycogen synthase kinase 3beta, protein kinase C, and proteinase-activated receptor-2 (PAR-2), which is consistent with the expression patterns of those proteins in human palmoplantar skin. Treatment of reconstructed skin with DKK1 reproduced the expression patterns of those key proteins observed in palmoplantar skin. These findings further elucidate why human skin is thicker and paler on the palms and soles than on the trunk through topographical and site-specific differences in the secretion of DKK1 by dermal fibroblasts that affects the overlying epidermis.