Recent work, especially in the yeast Saccharomyces cerevisiae, has demonstrated that mRNA movement from active translation to cytoplasmic granules, termed mRNA processing bodies (“p-bodies”), occurs in concert with the regulation of translation during cell stress. However, the factors regulating p-body formation are poorly defined. Recent data demonstrated a function for sphingolipids in regulating translation during heat stress, which led to the current hypothesis that p-bodies may form during heat stress in a sphingolipid-dependent manner. In this study, we demonstrate that heat stress induced formation of p-bodies as determined by localization of a GFP-tagged Dcp2p and RFP-tagged Edc3p to discrete cytoplasmic foci. Sphingoid base synthesis was required for this effect, in that inhibition of sphingoid base synthesis attenuated formation of these foci during heat stress. Moreover, treatment of yeast with exogenous sphingoid bases phyto- and dihydrosphingosine promoted formation of p-bodies in the absence of heat stress, and the lcb4/5 double deletion yeast mutant defective in sphingoid base kinase activity demonstrated both highly elevated sphingoid bases and large, clearly defined p-bodies under non-stress conditions. Functionally, inhibition of sphingolipid synthesis during heat did not prevent translation arrest but extended translation arrest, supporting a role for sphingoid bases in the resumption of translation following heat stress. Together, these data demonstrate a critical and novel role for sphingolipids in mediating p-body formation during heat. While roles of p-bodies in translation are the subject of much current investigation, these data indicate a novel function for sphingoid bases in p-body formation during heat stress and may help to elucidate mechanisms by which sphingolipids regulate translation.
Accumulation of aggregated forms of αSyn (α-synuclein) into Lewy bodies is a known hallmark associated with neuronal cell death in Parkinson's disease. When expressed in the yeast Saccharomyces cerevisiae, αSyn interacts with the plasma membrane, forms inclusions and causes a concentration-dependent growth defect. We have used a yeast mutant, cog6Δ, which is particularly sensitive to moderate αSyn expression, for screening a mouse brain-specific cDNA library in order to identify mammalian proteins that counteract αSyn toxicity. The mouse ribosomal and chaperone protein RPS3A was identified as a suppressor of αSyn [WT (wild-type) and A53T] toxicity in yeast. We demonstrated that the 50 N-terminal amino acids are essential for this function. The yeast homologues of RPS3A were not effective in suppressing the αSyn-induced growth defect, illustrating the potential of our screening system to identify modifiers that would be missed using yeast gene overexpression as the first screening step. Co-expression of mouse RPS3A delayed the formation of αSyn–GFP inclusions in the yeast cells. The results of the present study suggest that the recently identified extraribosomal chaperonin function of RPS3A also acts on the neurodegeneration-related protein αSyn and reveal a new avenue for identifying promising candidate mammalian proteins involved in αSyn functioning.
A yeast screening system was developed and successfully used to isolate a brain protein that counteracts the toxic effects of the main protein involved in Parkinson's disease.
component of oligomeric Golgi complex 6 (COG6); Parkinson’s disease; ribosomal and chaperone protein S3A (RPS3A); α-synuclein; yeast screening; COG6, component of oligomeric Golgi complex 6; ER, endoplasmic reticulum; 5-FOA, 5-fluoro-orotic acid; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; HBx, hepatitis B protein X; HRP, horseradish peroxidase; LB, Lewy body; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NA, numerical aperture; PD, Parkinson’s disease; Pgk1, phosphoglycerate kinase 1; PSMA2, proteasome subunit α type 2; Spir1CT, C-terminus of Spire homologue 1; RPS3A, ribosomal protein S3A; SC, synthetic complete; SD, synthetic dextrose; SNARE, soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor; αSyn, α-synuclein; WT, wild-type; yeGFP, yeast-enhanced GFP; YPD, yeast extract/peptone/dextrose
We show that highly efficient depletion of sphingolipids in two different cell lines does not abrogate the ability to isolate Lubrol-based DRMs (detergent-resistant membranes) or detergent-free lipid rafts from these cells. Compared with control, DRM/detergent-free lipid raft fractions contain equal amounts of protein, cholesterol and phospholipid, whereas the classical DRM/lipid raft markers Src, caveolin-1 and flotillin display the same gradient distribution. DRMs/detergent-free lipid rafts themselves are severely depleted of sphingolipids. The fatty acid profile of the remaining sphingolipids as well as that of the glycerophospholipids shows several differences compared with control, most prominently an increase in highly saturated C16 species. The glycerophospholipid headgroup composition is unchanged in sphingolipid-depleted cells and cell-derived detergent-free lipid rafts. Sphingolipid depletion does not alter the localization of MRP1 (multidrug-resistance-related protein 1) in DRMs/detergent-free lipid rafts or MRP1-mediated efflux of carboxyfluorescein. We conclude that extensive sphingolipid depletion does not affect lipid raft integrity in two cell lines and does not affect the function of the lipid-raft-associated protein MRP1.
caveolin; detergent-free lipid raft; flotillin; multidrug-resistance-related protein 1 (MRP1); Neuro-2a cell; Src
Hepatic GK (glucokinase) plays a key role in maintaining glucose homoeostasis. Many stimuli regulate GK activity by controlling its gene transcription. We hypothesized that endogenous lipophilic molecules modulate hepatic Gck expression. Lipophilic molecules were extracted from rat livers, saponified and re-constituted as an LE (lipophilic extract). LE synergized with insulin to induce primary hepatocyte, but not β-cell, Gck expression in an SREBP-1c (sterol-regulatory-element-binding protein-1c)-independent manner. The dramatic induction of Gck mRNA resulted in a significant increase in GK activity. Subsequently, the active molecules were identified as retinol and retinal by MS after the purification of the active LE fractions. Retinoids synergized with insulin to induce Gck expression by the activation of both RAR [RA (retinoic acid) receptor] and RXR (retinoid X receptor). Inhibition of RAR activation completely abolished the effect of retinal. The hepatic GK specific activity and Gck mRNA levels of Zucker lean rats fed with a VAD [VA (vitamin A)-deficient] diet were significantly lower than those of rats fed with VAS (VA-sufficient) diet. Additionally, the hepatic Gck mRNA expression of Sprague–Dawley rats fed with a VAD diet was lower than that of rats fed with VA-marginal, -adequate or -supplemented diets. The reduced expression of Gck mRNA was increased after an intraperitoneal dose of RA in VAD rats. Furthermore, an intravenous injection of RA rapidly raised hepatic Gck expression in rats fed with a VAS control diet. Understanding the underlying mechanism that mediates the synergy may be helpful for developing a treatment strategy for patients with diabetes.
hepatic glucokinase gene expression; insulin; primary hepatocyte; retinoid; synergistic effect; vitamin A deficiency
Sphingosine 1-phosphate (S1P) is a pleiotropic lipid mediator involved in numerous cellular and physiological functions. Notable among these are cell survival and migration as well as lymphocyte trafficking. S1P, which exerts its effects via five G protein coupled receptors (S1P1-5), is formed by the action of two sphingosine kinases (SphKs). While SphK1 is the more intensively studied isotype, SphK2 is unique in it nuclear localization and has been reported to oppose some of the actions ascribed to SphK1. While several scaffolds of SphK1 inhibitors have been described, there is a scarcity of selective SphK2 inhibitors that are necessary to evaluate the downstream effects of inhibition of this isotype. Herein we report a cationic amphiphilic small molecule that is a selective SphK2 inhibitor. In the course of characterizing this compound in wild type and SphK null mice we discovered that administration of the inhibitor to wild type mice resulted in a rapid increase in blood S1P, which is in contrast to our SphK1 inhibitor that drives circulating S1P levels down. Using a cohort of F2 hybrid mice, we confirmed, compared to wild type mice, that circulating S1P levels were higher in SphK2 null mice and lower in SphK1 null mice. Thus both SphK1 and SphK2 inhibitors recapitulate the blood S1P levels observed in the corresponding null mice. Moreover, circulating S1P levels mirror SphK2 inhibitor levels providing a convenient biomarker of target engagement.
Sphingosine Kinase (SphK); Sphingosine 1-phosphate (S1P); SphK inhibitor
GSK3β (glycogen synthase kinase 3β) is a ubiquitous kinase that plays a key role in multiple intracellular signalling pathways, and increased GSK3β activity is implicated in disorders ranging from cancer to Alzheimer’s disease. In the present study, we provide the first evidence of increased hypothalamic signalling via GSK3β in leptin-deficient Lepob/ob mice and show that intracerebroventricular injection of a GSK3β inhibitor acutely improves glucose tolerance in these mice. The beneficial effect of the GSK3β inhibitor was dependent on hypothalamic signalling via PI3K (phosphoinositide 3-kinase), a key intracellular mediator of both leptin and insulin action. Conversely, neuron-specific overexpression of GSK3β in the mediobasal hypothalamus exacerbated the hyperphagia, obesity and impairment of glucose tolerance induced by a high-fat diet, while having little effect in controls fed standard chow. These results demonstrate that increased hypothalamic GSK3β signalling contributes to deleterious effects of leptin deficiency and exacerbates high-fat diet-induced weight gain and glucose intolerance.
adeno-associated virus; arcuate nucleus; Dickkopf 1; food intake; high-fat diet; obesity; synapsin; Type II diabetes
Genes involved in normal developmental processes attract attention as mediators of tumour progression as they facilitate migration of tumour cells. EMT (epithelial–mesenchymal transition), an essential part of embryonic development, tissue remodelling and wound repair, is crucial for tumour metastasis. Previously, zinc transporter ZIP6 [SLC39A6; solute carrier family 39 (zinc transporter), member 6; also known as LIV-1) was linked to EMT in zebrafish gastrulation through a STAT3 (signal transducer and activator of transcription 3) mechanism, resulting in nuclear localization of transcription factor Snail. In the present study, we show that zinc transporter ZIP6 is transcriptionally induced by STAT3 and unprecedented among zinc transporters, and is activated by N-terminal cleavage which triggers ZIP6 plasma membrane location and zinc influx. This zinc influx inactivates GSK-3β (glycogen synthase kinase 3β), either indirectly or directly via Akt or GSK-3β respectively, resulting in activation of Snail, which remains in the nucleus and acts as a transcriptional repressor of E-cadherin (epithelial cadherin), CDH1, causing cell rounding and detachment. This was mirrored by ZIP6-transfected cells which underwent EMT, detached from monolayers and exhibited resistance to anoikis by their ability to continue proliferating even after detachment. Our results indicate a causative role for ZIP6 in cell motility and migration, providing ZIP6 as a new target for prediction of clinical cancer spread and also suggesting a ZIP6-dependent mechanism of tumour metastasis.
We demonstrate a novel mechanism for the ability of cellular zinc to drive cell detachment and migration with implications for breast cancer spread. This mechanism involves a zinc uptake channel ZIP6 (also known as SLC39A6) and a transcription factor, STAT3.
breast cancer; cell detachment; epithelial–mesenchymal transition (EMT); LIV-1; signal transducer and activator of transcription 3 (STAT3); solute carrier family 39; member 6 (SLC39A6); ZIP6; CHO, Chinese-hamster ovary; CT, threshold cycle value; E-cadherin, epithelial cadherin; EGF, epidermal growth factor; ELP2, elongation protein 2; EMT, epithelial–mesenchymal transition; ER, oestrogen receptor; FAS, fulvestrant; GSK, glycogen synthase kinase; qPCR, quantitative real-time PCR; SC, cytoplasmic loop between TM3 and TM4; SLC39, solute carrier family 39; STAT3, signal transducer and activator of transcription 3; STATIP1, STAT3-interacting protein 1; TAM, 4-hydroxytamoxifen; TGF, transforming growth factor; TM, transmembrane; β-TrCP, β-transducing repeat-containing protein; ZIP, Zrt- and Irt-like proteins
Pro-inflammatory cytokines and growth factors such as vascular endothelial growth factor (VEGF) contribute to the loss of the blood-retinal barrier (BRB) and subsequent macular edema in various retinal pathologies. VEGF signaling requires conventional PKC (PKCβ) activity; however, PKCβ inhibition only partially prevents VEGF-induced endothelial permeability and does not affect pro-inflammatory cytokine-induced permeability suggesting the involvement of alternative signaling pathways. Here, we provide evidence for the involvement of atypical protein kinase C (aPKC) signaling in VEGF-induced endothelial permeability and identify a novel class of inhibitors of aPKC that prevent BRB breakdown in vivo. Genetic and pharmacological manipulations of aPKC isoforms were used to assess their contribution to endothelial permeability in culture. A chemical library was screened using an in vitro kinase assay to identify novel small molecule inhibitors and further medicinal chemistry was performed to delineate a novel pharmacophore. We demonstrate that aPKC isoforms are both sufficient and required for VEGF-induced endothelial permeability. Furthermore, these specific, potent, non-competitive, small molecule inhibitors prevented VEGF-induced tight junction internalization and retinal endothelial permeability in response to VEGF in both primary culture and in rodent retina. These data suggest that aPKC inhibition with 2-amino-4-phenyl-thiophene derivatives may be developed to preserve the BRB in retinal diseases such as diabetic retinopathy or uveitis and the blood-brain barrier (BBB) in the presence of brain tumors.
vascular endothelial growth factor (VEGF); atypical protein kinase C (aPKC); blood-retinal barrier (BRB); blood-brain barrier (BBB)
Gene expression in eukaryotes is dependent on the mRNA methyl cap which mediates mRNA processing and translation initiation. Synthesis of the methyl cap initiates with the addition of 7-methylguanosine to the initiating nucleotide of RNA pol II (polymerase II) transcripts, which occurs predominantly during transcription and in mammals is catalysed by RNGTT (RNA guanylyltransferase and 5′ phosphatase) and RNMT (RNA guanine-7 methyltransferase). RNMT has a methyltransferase domain and an N-terminal domain whose function is unclear; it is conserved in mammals, but not required for cap methyltransferase activity. In the present study we report that the N-terminal domain is necessary and sufficient for RNMT recruitment to transcription initiation sites and that recruitment occurs in a DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole)-dependent manner. The RNMT-activating subunit, RAM (RNMT-activating miniprotein), is also recruited to transcription initiation sites via an interaction with RNMT. The RNMT N-terminal domain is required for transcript expression, translation and cell proliferation.
The mRNA methyl cap recruits the mediators of processing events and translation initiation. We report that RNMT, the human cap methyltransferase, is recruited to RNA polymerase II via the N-terminal domain and is required for gene expression and cell proliferation.
gene expression; methyl cap; transcription; translation; CTD, C-terminal domain; DMEM, Dulbecco's modified Eagle's medium; DRB, 5,6-dichloro-1-β-D-ribofuranosyl benzimidazole; GST, glutathione transferase; HA, haemagglutinin; IMEC, immortalized mammary epithelial cell; HEK, human embryonic kidney; NLS, nuclear localization signal; pol II, polymerase II; RAM, RNMT-activating miniprotein; RNGTT, RNA guanylyltransferase and 5′ phosphatase; RNMT, RNA guanine-7 methyltransferase; TSS, transcriptional start site; WT, wild-type
Many bacteria have evolved ways to interact with glycosylation functions of the immune system of their hosts. Streptococcus pyogenes [GAS (group A Streptococcus)] secretes the enzyme EndoS that cleaves glycans on human IgG and impairs the effector functions of the antibody. The ndoS gene, encoding EndoS, has, until now, been thought to be conserved throughout the serotypes. However, in the present study, we identify EndoS2, an endoglycosidase in serotype M49 GAS strains. We characterized EndoS2 and the corresponding ndoS2 gene using sequencing, bioinformatics, phylogenetic analysis, recombinant expression and LC–MS analysis of glycosidic activity. This revealed that EndoS2 is present exclusively, and highly conserved, in serotype M49 of GAS and is only 37% identical with EndoS. EndoS2 showed endo-β-N-acetylglucosaminidase activity on all N-linked glycans of IgG and on biantennary and sialylated glycans of AGP (α1-acid glycoprotein). The enzyme was found to act only on native IgG and AGP and to be specific for free biantennary glycans with or without terminal sialylation. GAS M49 expression of EndoS2 was monitored in relation to carbohydrates present in the culture medium and was linked to the presence of sucrose. We conclude that EndoS2 is a unique endoglycosidase in serotype M49 and differs from EndoS of other GAS strains by targeting both IgG and AGP. EndoS2 expands the repertoire of GAS effectors that modify key glycosylated molecules of host defence.
In this study, the endoglycosidase EndoS2 was characterized. The enzyme was found to be unique and conserved in serotype M49 of group A Streptococcus and to specifically cleave N-linked glycans on IgG and AGP.
α1-acid glycoprotein; endo-β-N-acetylglucosaminidase; host–pathogen interaction; IgG glycosylation; Streptococcus pyogenes; 2-AB, 2-aminobenzamide; ABS, Arthrobacter ureafaciens sialidase; AGP, α1-acid glycoprotein; AMF, almond meal α-fucosidase; BEH, bridged ethane–silicon hybrid; BKF, bovine kidney α-fucosidase; BTG, bovine testes β-galactosidase; CM, C-medium; CcpA, catabolite control protein A; FcγR, Fcγ receptor; FLD, fluorescence detection; GAS, group A Streptococcus; GH18, family 18 of glycoside hydrolases; HILIC, hydrophilic interaction liquid chromatography; HRP, horseradish peroxidase; LCA, Lens culinaris agglutinin; 4MU-GlcNAc, 4-methylumbelliferyl N-acetyl-β-D-glucosaminide; MWCO, molecular-mass cut-off; NAN1, neuraminidase/sialidase 1; PNGase F, peptide N-glycosidase F; r, recombinant; UHPLC, ultra-HPLC
The resistance of mosquitoes to chemical insecticides is threatening vector control programmes worldwide. Cytochrome P450 monooxygenases (CYPs) are known to play a major role in insecticide resistance, allowing resistant insects to metabolize insecticides at a higher rate. Among them, members of the mosquito CYP6Z subfamily, like Aedes aegypti CYP6Z8 and its Anopheles gambiae orthologue CYP6Z2, have been frequently associated with pyrethroid resistance. However, their role in the pyrethroid degradation pathway remains unclear. In the present study, we created a genetically modified yeast strain overexpressing Ae. aegypti cytochrome P450 reductase and CYP6Z8, thereby producing the first mosquito P450–CPR (NADPH-cytochrome P450-reductase) complex in a yeast recombinant system. The results of the present study show that: (i) CYP6Z8 metabolizes PBAlc (3-phenoxybenzoic alcohol) and PBAld (3-phenoxybenzaldehyde), common pyrethroid metabolites produced by carboxylesterases, producing PBA (3-phenoxybenzoic acid); (ii) CYP6Z8 transcription is induced by PBAlc, PBAld and PBA; (iii) An. gambiae CYP6Z2 metabolizes PBAlc and PBAld in the same way; (iv) PBA is the major metabolite produced in vivo and is excreted without further modification; and (v) in silico modelling of substrate–enzyme interactions supports a similar role of other mosquito CYP6Zs in pyrethroid degradation. By playing a pivotal role in the degradation of pyrethroid insecticides, mosquito CYP6Zs thus represent good targets for mosquito-resistance management strategies.
The key role of mosquito cytochrome P450 monooxygenases from the CYP6Z subfamily in the resistance of mosquitoes to insecticides was revealed. By using functional expression and in silico modelling, the capacity of CYP6Zs to degrade insecticide metabolites produced by esterase-mediated hydrolysis was demonstrated.
cytochrome P450 monooxygenase; insecticide; metabolism; mosquito; pyrethroid; recombinant system; resistance; AeCPR, Aedes aegypti CPR; CPR, NADPH-cytochrome P450-reductase; Cyt b5, cytochrome b5; 7-OH, 7-hydroxycoumarin; PBA, 3-phenoxybenzoic acid; PBAlc, 3-phenoxybenzoic alcohol; PBAld, 3-phenoxybenzaldehyde; qPCR, quantitative real-time PCR; Rt, retention time; SRS, substrate recognition site; TFA, trifluoroacetic acid
The LRRK2 (leucine-rich repeat protein kinase-2) is mutated in a significant number of Parkinson’s disease patients, but little is known about its regulation and function. A common mutation changing Gly2019 to serine enhances catalytic activity, suggesting that small-molecule inhibitors might have utility in treating Parkinson’s disease. We employed various approaches to explore the substrate-specificity requirements of LRRK2 and elaborated a peptide substrate termed Nictide, that had 20-fold lower Km and nearly 2-fold higher Vmax than the widely deployed LRRKtide substrate. We demonstrate that LRRK2 has marked preference for phosphorylating threonine over serine. We also observed that several ROCK (Rho kinase) inhibitors such as Y-27632 and H-1152, suppressed LRRK2 with similar potency to which they inhibited ROCK2. In contrast, GSK429286A, a selective ROCK inhibitor, did not significantly inhibit LRRK2. We also identified a mutant LRRK2[A2016T] that was normally active, but resistant to H-1152 and Y-27632, as well as sunitinib, a structurally unrelated multikinase inhibitor that, in contrast with other compounds, suppresses LRRK2, but not ROCK. We have also developed the first sensitive antibody that enables measurement of endogenous LRRK2 protein levels and kinase activity as well as shRNA (short hairpin RNA) methods to reduce LRRK2 expression. Finally, we describe a pharmacological approach to validate whether substrates are phosphorylated by LRRK2 and use this to provide evidence that LRRK2 may not be rate-limiting for the phosphorylation of the proposed substrate moesin. The findings of the present study will aid with the investigation of LRRK2.
leucine-rich repeat protein kinase-2 (LRRK2); moesin; Parkinson’s disease; phosphorylation; Rho kinase (ROCK)
Kidney function declines with advancing age and mitochondria have been implicated. We have examined the integrated function of mitochondria isolated from kidneys of 6 and 24 month Fischer 344 rats. Oxidative phosphorylation (OXPHOS) of intact mitochondria and cytochrome c oxidase activity in permeabilized mitochondria were determined with polarographic assays. The activities of the electron transport chain (ETC) complexes and the cytochrome content in solubilized mitochondria were measured using spectrophotometric methods. The respiratory complexes were evaluated with blue-native gel electrophoresis. Mitochondrial preparations were evaluated by immunoblotting for cytochrome c, Smac/Diablo, and the voltage dependant ion channel (VDAC). Mitochondrial morphology was examined by electron microscopy. OXPHOS of mitochondria isolated from 24 month animals was decreased 15–25% with complex I, II, III, IV and fatty acid substrates. The electron microscopic appearance of mitochondria, the activity of the ETC complexes and the protein abundance of individual complexes and supercomplexes were unchanged. The content of cytochrome c was decreased by 37% in aged mitochondria as determined by spectrophotometric methods and confirmed with immunoblotting. Polarographic determination of cytochrome c oxidase activity with endogenous cytochrome c demonstrated a 23% reduction in aged mitochondria, which was corrected with the addition of exogenous cytochrome c. Renal mitochondrial OXPHOS decreased with aging in the Fischer 344 rat. Decreased mitochondrial cytochrome c content is a major factor contributing to the OXPHOS defect of mitochondria isolated from kidneys of elderly animals.
Aging; mitochondria; oxidative phosphorylation; electron transport chain; cytochrome c; cytochrome c oxidase
A critical cis-regulatory element for the cystic fibrosis transmembrane conductance regulator (CFTR) gene is located in intron 11, 100 kb distal to the promoter, with which it interacts. This sequence contains an intestine-selective enhancer and associates with enhancer signature proteins, such as p300, in addition to tissue-specific transcription factors (TFs). Here we identify critical TFs that are recruited to this element and demonstrate their importance in regulating CFTR expression. In vitro DNase I footprinting and electrophoretic mobility shift assays (EMSA) identified four cell-type selective regions that bound TFs in vitro. Chromatin immunoprecipitation (ChIP) identified forkhead box A1/A2 (FOXA1/A2), hepatocyte nuclear factor 1 (HNF1), and caudal type homeobox 2 (CDX2) as in vivo trans-interacting factors. Mutation of their binding sites in the intron 11 core compromised its enhancer activity when measured by reporter gene assay. Moreover, siRNA-mediated knockdown of CDX2 caused a significant reduction in endogenous CFTR transcription in intestinal cells, suggesting that this factor is critical for the maintenance of high levels of CFTR expression in these cells. The ChIP data also demonstrate that these TFs interact with multiple cis-regulatory elements across the CFTR locus implicating a more global role in intestinal expression of the gene.
Gene expression; transcriptional networks; enhancer; cis-acting regulatory elements; CFTR
Malaria, caused by Plasmodia parasites, affects hundreds of millions of people. As purine auxotrophs, Plasmodia use transporters to import host purines for subsequent metabolism by the purine salvage pathway. Thus, purine transporters are attractive drug targets. All sequenced Plasmodia genomes encode four Equilibrative Nucleoside Transporters (ENTs). During the pathogenic intraerythrocytic stages, ENT1 is a major route of purine nucleoside/nucleobase transport. Another plasma membrane purine transporter exists because Plasmodium falciparum ENT1-knockout parasites survive at supraphysiological purine concentrations. The other three ENTs have not been characterized functionally. Codon-optimized P. falciparum and P. vivax ENT4 were expressed in Xenopus laevis oocytes and substrate transport determined with radiolabeled substrates. ENT4 transported adenine and 2′-deoxyadenosine at the fastest rate, with millimolar-range apparent affinity. ENT4-expressing oocytes did not accumulate hypoxanthine, a key purine salvage pathway substrate, or AMP. Micromolar concentrations of the plant hormone cytokinin compounds inhibited both Pf- and Pv-ENT4. In contrast to PfENT1, ENT4 interacted with the Immucillin compounds in the millimolar range and was inhibited by 10 μM dipyridamole. Thus, ENT4 is a purine transporter with unique substrate and inhibitor specificity. Its role in parasite physiology remains uncertain but is likely significant because of the strong conservation of ENT4 homologues in Plasmodia genomes.
Plasmodium; adenine; adenosine; immucillin; metabolism
The mitochondrion-associated protein LRPPRC (leucine-rich pentatricopeptide repeat-containing) interacts with one of the microtubule-associated protein family members MAP1S (microtubule-associated protein 1 small form), originally named C19ORF5 (chromosome 19 open reading frame 5), to form a complex. MAP1S interacts with LC3 (light chain 3), the mammalian homologue of yeast autophagy marker ATG8 and one of the most important autophagy markers in mammalian cells, and helps the attachment of autophagosomes with microtubules for trafficking and recruitment of substrate mitochondria into autophagosomes for degradation. MAP1S activates autophagosomal biogenesis and degradation to remove misfolded/aggregated proteins and dysfunctional organelles such as mitochondria and suppress oxidative stress-induced genomic instability and tumorigenesis. Previously, various studies have attributed LRPPRC nucleic acid-associated functions. Instead, in the present study, we show that LRPPRC associates with mitochondria, interacts with Beclin 1 and Bcl-2 and forms a ternary complex to maintain the stability of Bcl-2. Suppression of LRPPRC leads to reduction in mitochondrial potential and reduction in Bcl-2. Lower levels of Bcl-2 lead to release of more Beclin 1 to form the Beclin 1–PI3KCIII (class III phosphoinositide 3-kinase) complex to activate autophagy and accelerate the turnover of dysfunctional mitochondria through the PI3K (phosphoinositide 3-kinase)/Akt/mTOR (mammalian target of rapamycin) pathway. The activation of autophagy induced by LRPPRC suppression occurs upstream of the ATG5–ATG12 conjugate-mediated conversion of LC3-I into LC3-II and has been confirmed in multiple mammalian cell lines with multiple autophagy markers including the size of GFP–LC3 punctate foci, the intensity of LC3-II and p62 protein and the size of the vacuolar structure. The activated autophagy enhances the removal of mitochondria through lysosomes. LRPPRC therefore acts to suppress the initiation of basal levels of autophagy to clean up dysfunctional mitochondria and other cellular debris during the normal cell cycle.
Mitochondrial protein LRPPRC interacts with LC3-interactive microtubule-associated MAP1S and regulates autophagy. It interacts with Beclin 1 and Bcl-2 to form a ternary complex to maintain Bcl-2 stability. LRPPRC suppression enriches the Beclin 1-PI3KCIII complex to activate autophagy and mitophagy.
ATG5; autophagy; Beclin 1; class III phosphoinositide 3-kinase (PI3KCIII); leucine-rich pentatricopeptide repeat-containing (LRPPRC); microtubule-associated protein 1 small form (MAP1S); mitochondrion; p27; AMPK, AMP-activated protein kinase; eIF4E, eukaryotic initiation factor 4E; HEK, human embryonic kidney; HRP, horseradish peroxidase; LAMP, lysosome-associated membrane protein; LC3, light chain 3; LRPPRC, leucine-rich pentatricopeptide repeat-containing; LSFC, Leigh syndrome, French-Canadian type; MAP1, microtubule-associated protein 1; MAP1S, MAP1 small form; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; PI3KCIII, class III PI3K; Tom20, translocase of the mitochondrial outer membrane 20; Vps, vacuolar protein sorting
PKNs form a subfamily of the AGC serine/threonine protein kinases, and have a catalytic domain homologous with that of PKC (protein kinase C) in the C-terminal region and three characteristic ACC (antiparallel coiled-coil) domain repeats in the N-terminal region. The preferred peptide phosphorylation motif for PKNs determined by a combinatorial peptide library method was highly similar to that of PKCs within a 10-amino-acid stretch. Previously reported PKN inhibitory compounds also inhibit PKCs to a similar extent, and no PKN selective inhibitors have been commercially available. We have identified a 15-amino-acid peptide inhibitor of PKNs based on amino acids 485–499 of the C-terminal region of the C2-like domain of PKN1. This peptide, designated as PRL, selectively inhibits the kinase activity of all isoforms of PKN (Ki = 0.7 μM) towards a peptide substrate, as well as autophosphorylation activity of PKN in vitro, in contrast with PKC. Reversible conjugation by a disulfide bond of a carrier peptide bearing a penetration accelerating sequence to PRL, facilitated the cellular uptake of this peptide and significantly inhibited phosphorylation of tau by PKN1 at the PKN1-specific phosphorylation site in vivo. This peptide may serve as a valuable tool for investigating PKN activation and PKN-mediated responses.
cell-penetrating peptide (CPP); penetration accelerating sequence octa-arginine (LPasR8); PRL; protein kinase C (PKC); PKN inhibitor
We previously revealed that tumor suppressor ATBF1 formed an autoregulatory feedback loop with estrogen-ERα signaling to regulate estrogen-dependent cell proliferation in breast cancer cells. In this loop, ATBF1 inhibits the function of estrogen-ERα signaling while ATBF1 protein levels are fine-tuned by estrogen-induced transcriptional upregulation as well as ubiquitin proteasome pathway (UPP)-mediated protein degradation. Here we show that the estrogen-responsive finger protein (EFP) is an E3 ubiquitin ligase mediating estrogen-induced ATBF1 protein degradation. Knockdown increases but overexpression of EFP decreases ATBF1 protein levels. EFP interacts with and ubiquitinates ATBF1 protein. Furthermore, we show that EFP is an important factor in estrogen-induced ATBF1 protein degradation in which some other factors are also involved. In human primary breast tumors, due to both as directly-upregulated ERα target gene products, the levels of ATBF1 protein are positively correlated with the levels of EFP protein. However, the ratio of ATBF1 protein to EFP protein is negatively correlated with EFP protein levels. Functionally, ATBF1 antagonizes EFP-mediated cell proliferation. These findings not only establish EFP as the E3 ubiquitin ligase for estrogen-induced ATBF1 protein degradation, but further support the autoregulatory feedback loop between ATBF1 and estrogen-ERα signaling and thus implicate ATBF1 in estrogen-dependent breast development and carcinogenesis.
Estrogen; ATBF1; Protein degradation; E3 ubiquitin ligase; EFP; Breast cancer
The covalent attachment of the small ubiquitin-like protein modifier (SUMO) to target proteins results in modifications in their activity, binding interactions, localization or half-life. The reversal of this modification is catalyzed by SUMO-specific processing proteases (SENPs). Mammals contain four SUMO paralogs and six SENP enzymes. Our studies describe a systematic analysis of human SENPs, integrating estimates of relative selectivity for SUMO1 and SUMO2, and kinetic measurements of recombinant C-terminal SENP catalytic domains (cSENPs). We first characterized the reaction of each endogenous SENP and their catalytic domains (cSENP) with HA-tagged SUMO1 and SUMO2 vinyl sulfones (HA-SUMO-VS), active site-directed irreversible inhibitors of SENPs. We found that all cSENPs and endogenous SENP1 react with both SUMO paralogs, while all other endogeneous SENPs in mammalian cells and tissues display high selectivity for SUMO2-VS. To obtain more quantitative data, the kinetic properties of purified cSENPs were determined using SUMO1 or SUMO2-amidomethyl coumarin (SUMO-AMC) as substrate. All enzymes bind their respective substrates with high affinity. cSENP1 and cSENP2 process either SUMO substrate with similar affinity and catalytic efficiency; cSENP5 and cSENP6 show marked catalytic specificity for SUMO2 as measured by KM and kcat while cSENP7 works only on SUMO2. Compared to cSENPs, recombinant full-length SENP1 and SENP2 show differences in SUMO selectivity indicating that paralog specificity is influenced by the presence of the variable N-terminal domain of each SENP. Our data suggests that SUMO2 metabolism is more dynamic than that of SUMO1 since most SENPs display a marked preference for SUMO2.
SUMO; paralogs; substrate specificity; SENP; desumoylation
The Prxs (peroxiredoxins) are a family of cysteine-dependent peroxidases that decompose hydrogen peroxide. Prxs become hyperoxidized when a sulfenic acid formed during the catalytic cycle reacts with hydrogen peroxide. In the present study, Western blot methodology was developed to quantify hyperoxidation of individual 2-Cys Prxs in cells. It revealed that Prx 1 and 2 were hyperoxidized at lower doses of hydrogen peroxide than would be predicted from in vitro data, suggesting intracellular factors that promote hyperoxidation. In contrast, mitochondrial Prx 3 was considerably more resistant to hyperoxidation. The concentration of Prx 3 was estimated at 125 μM in the mitochondrial matrix of Jurkat T-lymphoma cells. Although the local cellular environment could influence susceptibility, purified Prx 3 was also more resistant to hyperoxidation, suggesting that despite having C-terminal motifs similar to sensitive eukaryote Prxs, other structural features must contribute to the innate resilience of Prx 3 to hyperoxidation.
hydrogen peroxide; mitochondria; peroxiredoxin; oxidative stress; sulfenic acid; thiol
The intramolecular disulfide bond in human Cu,Zn superoxide dismutase 1 (hSOD1) plays a key role in maintaining the protein’s stability and quaternary structure. In mutant forms of SOD1 that cause familial amyotrophic lateral sclerosis (ALS), this disulfide bond is more susceptible to chemical reduction, which may lead to destabilization of the dimer and aggregation. During hSOD1 maturation, disulfide formation is catalyzed by the copper chaperone CCS1. Previous studies in yeast demonstrate that the yeast glutathione (GSH)/glutaredoxin redox system promotes reduction of the hSOD1 disulfide in the absence of CCS1. Herein, we further probe the interaction between hSOD1, GSH, and glutaredoxins to provide mechanistic insight into the redox kinetics and thermodynamics of the hSOD1 disulfide. We demonstrate that human glutaredoxin 1 (hGrx1) uses a monothiol mechanism to reduce the hSOD1 disulfide, and the GSH/hGrx1 system reduces ALS mutant SOD1 at a faster rate than WT hSOD1. However, redox potential measurements demonstrate that the thermodynamic stability of the disulfide is not consistently lower in ALS mutants compared to WT hSOD1. Furthermore, the presence of the metal cofactors does not influence the disulfide redox potential. Overall, these studies suggest that differences in the GSH/hGrx1 reaction rate with WT vs. ALS mutant hSOD1 and not the inherent thermodynamic stability of the hSOD1 disulfide bond may contribute to the greater pathogenicity of ALS mutant hSOD1.
Disulfide; redox potential; glutaredoxins; yeast; Cu,Zn superoxide dismutase; monothiol
mast cells; extracellular traps; HIF-1α; Staphylococcus aureus; innate immunity; antimicrobial
Silencing of GATA5 gene expression as a result of promoter hypermethylation has been observed in lung, gastrointestinal, and ovarian cancers. However, the regulation of GATA5 gene expression has been poorly understood. Here, we demonstrate that an enhancer (E)-box in the GATA5 promoter (bp −118 to −113 in mouse; bp −164 to −159 in the human) positively regulates GATA5 transcription by binding upstream stimulatory factor 1 (USF1). Using site-directed mutagenesis, EMSA, and affinity chromatography, we found that USF1 specifically binds to the E-box sequence (5′-CACGTG-3′), but not to a mutated E-box. CpG methylation of this E-box significantly diminished its binding of transcription factors. Mutation of the E-box within a GATA5 promoter fragment significantly decreased promoter activity in a luciferase reporter assay. Chromatin immunoprecipitation identified that USF1 physiologically interacts with the GATA5 promoter E-box in mouse intestinal mucosa, which has the highest GATA5 gene expression in mouse. Co-transfection with USF1 expression plasmid significantly increased GATA5 promoter-driven luciferase transcription. Furthermore, real-time and RT-PCR analyses confirmed that overexpression of USF1 activates endogenous GATA5 gene expression in human bronchial epithelial cells. This study presents the first evidence that USF1 activates GATA5 gene expression through E-box motif, and suggests a potential mechanism (disruption of the E-box) by which GATA5 promoter methylation reduces GATA5 expression in cancer.
differentiation; transcription factor; cancer
Obesity is associated with induction of endoplasmic reticulum (ER)-stress response signalling and insulin resistance. Protein tyrosine phosphatase (PTP)-1B is a major regulator of adiposity and insulin sensitivity. The aim of this study was to investigate the role of liver-PTP1B in chronically- (high-fat diet) and pharmacologically-induced (tunicamycin, thapsigargin) ER-stress response signalling in vitro and in vivo.
We assessed the effects of ER-stress response induction on hepatic PTP1B expression, and consequences of hepatic-PTP1B deficiency, in cells and mouse liver, on components of ER-stress response signalling.
We found that PTP1B protein and mRNA expression levels were up-regulated in response to acute and/or chronic ER-stress, in vitro and in vivo. Silencing PTP1B in hepatic cell lines or mouse liver (L-PTP1B−/−) protected against induction of pharmacologically- and/or obesity-induced ER-stress. High-fat diet-induced increase in CHOP and BIP mRNA levels were partially inhibited, whereas ATF4, GADD34, GRP94, ERDJ4 mRNAs and ATF6 protein cleavage were completely suppressed in L-PTP1B−/− mice relative to control littermates. L-PTP1B−/− mice also had increased nuclear translocation of spliced XBP-1 via increased p85α binding. We demonstrate that the ER-stress response and liver-PTP1B expression are interlinked in obesity and pharmacologically-induced ER-stress and this may be one of the mechanisms behind improved insulin sensitivity and lower lipid accumulation in L-PTP1B−/− mice.
ER stress; PTP1B; insulin resistance; obesity; metabolic syndrome
Hepatic glucose metabolism is strongly influenced by oxidative stress and pro-inflammatory stimuli. PON2 (paraoxonase 2), an enzyme with undefined antioxidant properties, protects against atherosclerosis. PON2-deficient (PON2-def) mice have elevated hepatic oxidative stress coupled with an exacerbated inflammatory response from PON2-deficient macrophages. In the present paper, we demonstrate that PON2 deficiency is associated with inhibitory insulin-mediated phosphorylation of hepatic IRS-1 (insulin receptor substrate-1). Unexpectedly, we observed a marked improvement in the hepatic IRS-1 phosphorylation state in PON2-def/apoE (apolipoprotein E)−/− mice, relative to apoE−/− mice. Factors secreted from activated macrophage cultures derived from PON2-def and PON2-def/apoE−/− mice are sufficient to modulate insulin signalling in cultured hepatocytes in a manner similar to that observed in vivo. We show that the protective effect on insulin signalling in PON2-def/apoE−/− mice is directly associated with altered production of macrophage proinflammatory mediators, but not elevated intracellular oxidative stress levels. We further present evidence that modulation of the macrophage inflammatory response in PON2-def/apoE−/− mice is mediated by a shift in the balance of NO and ONOO− (peroxynitrite) formation. Our results demonstrate that PON2 plays an important role in hepatic insulin signalling and underscores the influence of macrophage-mediated inflammatory response on hepatic insulin sensitivity.
apolipoprotein E (apoE); insulin receptor substrate-1 (IRS-1); liver; macrophage; paraoxonase; paraoxonase 2 (PON2)