Nrf2 plays a role in protection of cells against oxidative stress and xenobiotic damage by regulating cytoprotective genes. In this study, we investigated the effect of Nrf2 on melanogenesis in normal human melanocytes (NHMCs). When NHMCs were transduced with a recombinant adenovirus expressing Nrf2, melanin synthesis was significantly decreased. Consistent with this result, overexpression of Nrf2 decreased the expression of tyrosinase and tyrosinase-related protein 1. The inhibitory effect of Nrf2 was reversed by overexpression of Keap1, an intracellular regulator of Nrf2. Interestingly, Nrf2 overexpression resulted in marked activation of PI3K/Akt signaling. Conversely, inhibition of PI3K activity by treatment with wortmannin reversed the depigmentary effects of Nrf2. Taken together, these results strongly suggest that Nrf2 negatively regulates melanogenesis by modulating the PI3K/Akt signaling pathway. Skin is continuously exposed to environmental insults, including toxic chemicals and ultraviolet (UV) radiation, which can induce the formation of reactive oxygen species (ROS). Since excessive oxidative stress can evoke many biological responses, such as cell senescence, severe cell damage and even neoplastic transformation, cells must develop protective mechanisms. One important defense system includes phase II detoxification enzymes, which are regulated by the transcription factor nuclear factor E2-related factor 2 (Nrf2) With regard to skin pigmentation, there is limited evidence linking Nrf2 to vitiligo. Vitiligo is an acquired pigmentary disorder of the skin and mucous membranes that is characterized by loss of pigmentation. For instance, Nrf2 promoter polymorphisms are associated with an increased risk of vitiligo Initially, we performed Western blotting to assess endogenous expression of Nrf2 and its intracellular regulator Keap1 in cultured human skin cells. We used primary cultured NHEMs, SV40T-transformed human epidermal keratinocytes (SV-HEKs) and normal human dermal fibroblasts (NHDFs). In all skin cells, expression of Nrf2 and Keap1 was detected by Western blotting .To investigate the effect of Nrf2, we created a recombinant adenovirus expressing Flag-tagged Nrf2. Adenoviral transduction did not affect the viability of NHEMs . After tTyrosinase is a rate-limiting enzyme for controlling the production of melanin, thus we examined the effect of Nrf2 overexpression on tyrosinase (TYR) activity. Consistent with previous data, Nrf2 overexpression led to inhibition of TYR activity compared to the control group  . SimilarWhen NHEMs were treated with Nrf2 inducers such as tert-butylhydroquinone (tBHQ), sulforaphane and curcumin, nuclear translocation of Nrf2 was observed . In lineAs Nrf2 activity is negatively regulated by Keap1, we next determined whether overexpression of Keap1 inhibited the effect of Nrf2 on melanogenesis. To this end, we co-transduced NHEMs with adenoviruses expressing Nrf2 and/or Keap1. First, we determined whether Keap1 could prevent the activation of Nrf2 in NHMCs. As Nrf2 activity is regulated by translocation to the nucleus, we assessed whether Keap1 could inhibit the nuclear translocation of Nrf2 in NHMCs. As expected, overexpression of Keap1 effectively prevented translocation of Nrf2 to the nucleus . Next, wWe further confirmed the negative role of Nrf2 on pigmentation by knockdown experiment. To this end, we transduced NHEMs with recombinant adenovirus expressing microRNA (miR) specific for Nrf2. As shown in It is known that melanogenesis is regulated by the balance between a variety of signal transduction pathways, including the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA), p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK), and phosphoinositide 3-kinase/Akt (PI3K/Akt) pathways To further confirm the involvement of the PI3K/Akt signaling pathway in Nrf2-induced depigmentation of melanocytes, we treated NHEMs with the PI3K inhibitor wortmannin. As a result, PI3K inhibitor treatment effectively blocked Nrf2-induced signaling activation. Concomitantly, Nrf2-induced repression of TYR and TRP1 protein expression was reversed by PI3K inhibitor treatment . FinallyIn this study, we demonstrated an inhibitory role for Nrf2 in melanogenesis using an adenoviral gene delivery system. Specifically, Nrf2 overexpression in NHEMs leads to significant inhibition of melanogenesis as a result of PI3K/Akt pathway activation, an inhibitory effect that is reversed by Keap1 overexpression.It was previously demonstrated that some Nrf2 inducers, such as tert-butylhydroquinone (tBHQ), sulforaphane and curcumin, have depigmentary effects. For example, repeated dermal application of tBHQ leads to depigmentation in black guinea pigs In our study, overexpression of Nrf2 in NHEMs resulted in downregulation of TYR and TRP1 gene expression. Although Nrf2 is known to be a transcription factor that can bind to cognate DNA sequences in target genes, it is unlikely that Nrf2 directly regulates the transcription of TYR and TRP1 at the genomic level. This speculation is based on the fact that there are no known Nrf2 binding sequences in the promoters of TYR and TRP1, and that activation of Nrf2 leads to downregulation of gene expression. Thus, we hypothesized that the effect of Nrf2 may be a secondary event following gene expression and/or may be linked to the modulation of intracellular signaling. Interestingly, we found that overexpression of Nrf2 led to a robust increase in the phosphorylation of Akt. Furthermore, several signaling molecules acting downstream of Akt, such as mTOR and S6 kinase, were also activated by Nrf2. These results strongly suggest that the mechanism underlying Nrf2-induced depigmentation is modulation of intracellular signaling. This speculation is supported by previous reports that the specific PI3K inhibitors wortmannin and LY294002 stimulate melanin synthesis From dermatological and cosmetic viewpoints, the development of depigmenting and/or whitening agents is still an important issue. In this context, efforts are still focused on the development of compounds that can inhibit TYR and/or the transcription factor MITF. We suggest that the Nrf2/Keap1 pathway may be a good target for whitening agents, because the skin protective role of Nrf2 has been demonstrated. That is, non-toxic Nrf2 inducers may be cosmetically and pharmaceutically beneficial because we can expect a dual effect of Nrf2 on skin protection from UV-induced skin damage and inhibition of pigmentation.In summary, we demonstrated a novel role for Nrf2 in the regulation of melanogenesis. Our results may contribute to a better understanding of the regulatory mechanisms of melanogenesis, and may help in the development of new targets for pigment-related skin diseases and hypopigmentation reagents.This study was approved by the Institutional Review Board of Chungnam National University School of Medicine. All human skin samples were obtained under the written informed consent of donors.Foreskin specimens were briefly sterilized in 70% ethanol, minced, and then treated with dispase for overnight at 4°C. The epidermis was separated and placed in a solution containing 0.05% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA)  for 30 min at 37°C. After vigorous pipetting, cells were pelleted and resuspended in growth medium, which is composed of Medium 254 and human melanocyte growth supplement . During the primary culture of human melanocytes, 200 µg/ml of G418  was added to the growth medium to suppress the proliferation of fibroblasts Total RNA was isolated from cultured skin epithelial cells using Easy-blue RNA extraction kit . Two μg of total RNA was reverse transcribed with moloney-murine leukaemia virus (M-MLV) reverse transcriptase . Aliquot of RT mixture was subjected to PCR cycles with primer set for Nrf2 , and Keap1 . The Nrf2 and Keap1 cDNAs were subcloned into pENTR/CMV-Flag vector that has attL sites for site-specific recombination with a Gateway destination vector . The replication-incompetent adenoviruses were created using Virapower adenovirus expression system (Invitrogen) according to the method previously described http://rnaidesigner.lifetechnologies.com/rnaiexpress). The double-stranded DNA oligonucleotides were synthesized and cloned into the parental vector pcDNA6.2-GW/EmGFP-miR (Invitrogen). The expression cassette for miR was moved into pENT/CMV vector, and then adenovirus was made. The miR sequences were as follows: Nrf2, top strand 5'- TGCTGTAGATCAGAAACATCAATGGGGTTTTGGCCACTGACTGACCCCATTGATTTCTGATCTA-3' and bottom strand 5'- CCTGTAGATCAGAAATCAATGGGGTCAGTCAGTGGCCAAAACCCCATTGATGTTTCTGATCTAC-3'; Akt1, top strand 5'- TGCTGTGGAAGGTGCGTTCGATGACAGTTTTGGCCACTGACTGACTGTCATCGCGCACCTTCCA-3' and bottom strand 5'- CCTGTGGAAGGTGCGCGATGACAGTCAGTCAGTGGCCAAAACTGTCATCGAACGCACCTTCCAC-3'.For miR specific for Nrf2 and Akt1, target sequences were designed using Invitrogen's RNAi Designer (Cells were harvested by centrifugation and then lysed in protein extraction solution (Intron). After vigorous pipetting, extracts were centrifuged for 15 min at 15,000 rpm. Total protein was measured using a BCA protein assay kit . Samples (20–30 µg protein per lane) were run on SDS-polyacrylamide gels, transferred onto nitrocellulose membranes and incubated with appropriate antibodies for overnight at 4°C with gentle agitation. Blots were then incubated with peroxidase-conjugated secondary antibodies for 30 minutes at room temperature, and visualized by enhanced chemiluminescence (Intron). The following primary antibodies were used in this study: Nrf2, Keap1, tyrosinase, TRP1, TRP2, actin and laminB , phospho Akt, total Akt, phospho mTOR, total mTOR and phospho S6 ribosomal proteins .Nuclear extracts from the cells were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific), according to the recommended protocol. To confirm the purity of subcellular fractionation, the extracts were Western blotted and probed with nuclear specific anti-laminB antibody.For determination of pellet pigmentation, cells were collected and pelleted by centrifugation. Melanin pigment was dissolved in 1 N NaOH at 100°C for 30 min, and quantified by measuring optical density at 405 nm. Tyrosinase activity was determined as described previously with slight modification Cells were grown at 50% confluency in 6-well culture plate, then co-transduced with adenovirus harboring TYR promoter reporter cassette and adenoviruses expressing Flag-tagged Nrf2 and/or Flag-tagged Keap1. After incubation for 6 h, cells were replenished with fresh medium. Cells were further incubated for 2 days. Luciferase activities were determined using Luciferase assay system , according to the recommended protocol.Data were evaluated statistically using one-way ANOVA using SPSS software (ver 22.0). Statistical significance was set at p<0.01.Figure S1Cell viability after adenoviral transduction. NHEMs were transduced with an adenovirus expressing Nrf2 or LacZ (control) at the indicated multiplicity of infection (MOI) for 6 h. Cells were replenished and then cultured for a further 3 days. Cell viability was determined by MTT assay.(PDF)Click here for additional data file.Figure S2NHEMs were transduced with an adenovirus expressing Nrf2 or LacZ (control) at the indicated multiplicity of infection (MOI) for 6 h. Cells were replenished and then cultured for a further 3 days. The mRNA levels were evaluated by RT-PCR. CYP (cyclophilin) was used as loading control.(PDF)Click here for additional data file.Figure S3NHEMs were transduced with adenovirus expressing microRNA-Nrf2 (miR-Nrf2) or scrambled control (miR-scr) at the 10 multiplicity of infection (MOIs) for 6 h. Cells were replenished and then cultured for a further 3 days. Effect of Nrf2 Knockdown on melanogenesis is determined by (A) Western blotting, (B) TYR activity and (C) TYR promoter activity. (*P<0.01 vs. control).(PDF)Click here for additional data file.Figure S4NHEMs were co-transduced with indicated adenoviruses at the 10 MOIs for 6 h. Cells were replenished and then cultured for a further 3 days. Expression of Nrf2 and pigmentation-related genes was determined by Western blot. Kockdown of Akt by miR significantly prevented Nrf2-induced downregulation of TYR and TRP1.(PDF)Click here for additional data file.Figure S5NHEMs were treated with Nrf2 inducers such as tert-butylhydroquinone (tBHQ), sulforaphane and curcumin, for the indicated time points. Total cell pellets were separated into cytosolic and nuclear fraction. Nrf2 translocation was determined by Western blot. To confirm the purity of subcellular fractionation, the extracts were probed with cytosol specific anti-α-tubulin and nucleus specific anti-laminB antibody. All experiments were performed three times. M: marker.(PDF)Click here for additional data file.Figure S6(A) NHEMs were treated with curcumin at the indicated concentrations for 3 days. Expression of Nrf2, tyrosinase (TYR), and phospho-Akt was determined by Western blot. (B) NHEMs were transduced with adenovirus expressing miR-Nrf2 or miR-scr. Cells were replenished and treated with curcumin (12.5 µM) for 3 days. TYR activity was determined and expressed as a percentage of the control. Data are the means ± SD of triplicate measurements (*P<0.01 vs. control).(PDF)Click here for additional data file.


Meleagris gallopavo) with gastrointestinal disease from a farm in Indiana. Its genome organization is as follows: 5′ untranslated region (UTR)IRES-II  3′ UTR-poly(A). TuASV shares only 34% (P1), 36% (P2), and 35% (P3) amino acid identities with avihepatoviruses, indicating that it potentially represents a novel picornavirus genus.A novel picornavirus, turkey avisivirus (TuASV), was identified from the feces of turkeys ( Picornaviridae consists of 12 formal genera, but potential new genera infecting humans and domestic and wild animals have been recently described (Picornaviridae website [http://www.picornaviridae.com]). Turkey production is an important food industry worldwide, valued at $5 billion in the United States in 2011  based on its relatedness to the duck hepatitis A virus of the genus Avihepatovirus, and for consistency with a publication in press describing a closely related virus named TuASV-1 (strain turkey/M176-TuASV/2011/HUN); TuASV-1 was found in Hungarian turkeys with stunting syndrome and/or poultry enteritis . The 5′ UTR was 609 bases long, containing a type II internal ribosome entry site (IRES-II), followed by a 6,495-base ORF  encoding the polyprotein P1, P2, and P3 regions. The 3′ UTR was 266 bases long, followed by poly(A). Both 2A proteins (2A2 and 2A3) and the 2C protein contained the helicase nucleotide-binding motif GXXGXGK(T/S). Surprisingly, the closely related Hungarian viruses contained a third 2A protein (2A1) that is missing in the U.S. strain.The virus was provisionally named turkey avisivirus , 36% (P2), and 35% (P3) amino acid identities with duck hepatitis virus (DHV) and lower identities with other picornaviruses. Based on the International Committee on Taxonomy of Viruses (ICTV) criteria, members of the same picornavirus genus share, for P1, P2, and P3 regions, >40%, 40%, and 50% amino acid identities, respectively. Therefore, TuASV-USA-IN1 likely reflects the presence of a new genus in the family KC614703.The complete genome sequence of TuASV-USA-IN1 has been deposited in GenBank under accession no.


It has been presumed that alkali metal ions (whether Na+ or K+) coordinated to chelating ligands can cross the hydrophobic cell membrane easily and can function effectively for depolarizing the ion difference. This unique function was utilized for bacterial cell death in which K+ has been found coordinated valinomycin (antibiotic).Enormous interest had been paid to the coordination chemistry of alkali and alkaline metal ions because of their role inside body viz; their Li1) with dicoumarol ligand, 4-Hydroxy-3-[-phenyl-methyl]-chromen-2-one (L) is isolated from the saturated solution of sodium methoxide. Single crystal X-ray diffraction studies of the adduct reveals that sodium is in the form of cation attached to a methoxide, methanol and a dicoumarol ligand where carbonyl functional groups of the coumarin derivative are acting as bridges. The sodium compound (1) is also characterized by IR, 1H-NMR, and 13C{1H}-NMR spectroscopic techniques. The composition is confirmed by elemental analysis. DFT study for 1 has been carried out using B3LYP/6-13G calculations which shown the theoretical confirmation of the various bond lengths and bond angles. Both the compounds were studied subsequently for the U2OS tumoricidal activity and it was found that L has LD50 value of 200 μM whereas the sodium analog cytotoxicity did not drop down below 60%.Distinct sodium adduct (1) with medicinally important dicoumarol ligand (L) has been reported. The crystal structure and DFT study confirm the formation of cationic sodium compound with dicoumarol. The ligand was found more active than the sodium analog attributed to the instability of 1 in solution state. Coumarin compound with sodium was observed to be less cytotoxic than the ligand, its LD50 value never dropped below 60%.A sodium analogue ( This work is dealing with the synthesis of sodium derivative of dicoumarols. The structure was assigned based upon the single crystal diffraction and DFT studies. It was presumed that the novel sodium compound will show very good results in biological system but the opposite behavior was observed owing to the instability of the compound in solution phase. We are searching for the possible answers in this unique research and soon will be available to the reader.In malignant sarcoma the neoplasm of the malignant tissues produces malignant osteoid which causes bone cancer. This is prevailing 4 in 100,000 people/year and is a leading cancer disease in children’s under age 15. Leg or hand amputation, a disaster to the human or animal body, is caused by osteosarcoma + (100), Elemental Analysis (C25H16O6), Calc. C: 72.81%, H:3.91% Exp. C: 72.83, H: 3.88, Λm: 240 μS.The dicoumarol ligand : 3658 (OH); 1671 (CO); 1534 (C = C); 1349 (Na-O lactone). 1H NMR  : 2.52 ; 6.10 ; 7.11-7.67 ; 8.31 ; 13C {1H}-NMR  : 33 (C6); 103 ; 106 (C19); 116 ; 117 ; 121 ; 122 ; 123 (C16); 131 ; 145 ; 164 ; 167 . 23Na-NMR  : -1.01. MS, m/ z (%): No molecular ion peak observed. Elemental Analysis (C54H44Na2O16), Calc. C: 65.19%, H: 4.46% Exp. C: 65.23%, H: 4.23, Λm: 410 μS.10 mmol of the dicoumarol compound was added to 20 cm1) with medicinally important dicoumarol ligand (L) has been reported. Spectral analysis and X-ray crystallographic techniques have been used to determine structure of the compound. The crystal structure and DFT study confirm the formation of cationic sodium compound with dicoumarol. The ligand was found more active than the sodium analog attributed to the instability of 1 in solution state. Coumarin compound with sodium was observed to be less cytotoxic than the ligand, its LD50 value never dropped below 60%.A sodium analogue (The authors declare that they have no competing interests.SR and MI carried out the original laboratory work in the lab. of RJB under the supervision of SUR. EA, SM and KHM analysed the samples for their U2OS anticancer studies. AK and TSH carried out the theoretical studies. The structure was solved by SR and AJB. All authors read and approved the final manuscript.Supporting Information.Click here for file


We have previously reported that the mitochondria inhibitor 3-nitropropionic acid (3-NP), induces the expression of DNA damage-regulated autophagy modulator1 (DRAM1) and activation of autophagy in rat striatum. Although the role of DRAM1 in autophagy has been previously characterized, the detailed mechanism by which DRAM1 regulates autophagy activity has not been fully understood. The present study investigated the role of DRAM1 in regulating autophagy flux. In A549 cells expressing wilt-type TP53, 3-NP increased the protein levels of DRAM1 and LC3-II, whereas decreased the levels of SQSTM1 (sequestosome 1). The increase in LC3-II and decrease in SQSTM1 were blocked by the autophagy inhibitor 3-methyl-adenine. Lack of TP53 or knock-down of TP53 in cells impaired the induction of DRAM1. Knock-down of DRAM1 with siRNA significantly reduced 3-NP-induced upregulation of LC3-II and downregulation of SQSTM1, indicating DRAM1 contributes to autophagy activation. Knock-down of DRAM1 robustly decreased rate of disappearance of induced autophagosomes, increased RFP-LC3 fluorescence dots and decreased the decline of LC3-II after withdraw of rapamycin, indicating DRAM1 promotes autophagy flux. DRAM1 siRNA inhibited lysosomal V-ATPase and acidification of lysosomes. As a result, DRAM1 siRNA reduced activation of lysosomal cathepsin D. Similar to DRAM1 siRNA, lysosomal inhibitors E64d and chloroquine also inhibited clearance of autophagosomes and activation of lysosomal cathapsin D after 3-NP treatment. These data suggest that DRAM1 plays important roles in autophagy activation induced by mitochondria dysfunction. DRAM1 affects autophagy through argument of lysosomal acidification, fusion of lysosomes with autophagosomes and clearance of autophagosomes. DRAM1 promotes autophagy flux by enhancing lysosomal acidification.+/+) and H1299 (TP53−/−) and Hela cell lines were purchased from Shanghai Institute of Biochemistry and Cell Biology in China, and were grown at 37°C in 5% CO2 in RPMI supplemented with 2 mmol/L L-glutamine and 10% FCS. Primary mouse embryonic fibroblasts (MEFs) were derived from p53 wt and p53 KO sibling embryos, and maintained with DMEM supplemented with 10% FCS and antibiotics. 3-NP (N5636), 3-MA (M9281), carbonyl cyanide m-chlorophenylhydrazone , ATP (A6559), chloroquine (C6628), E-64d (E8640) and Z-Vad-FMK (V116) were all purchased from Sigma-Aldrich . LysoTracker Red (L7528) and LysoSentor (L7533) were purchased from Invitrogen-Molecular Probes . All cell culture reagents were purchased from Gibco  unless otherwise noted.A549 . The presence of several intense fluorescent dots in cells is indicative of the accumulation of autophagosomes. Transfection of cells with expression plasmids was performed using Lipofectamine 2000 . For each condition, the number of GFP-LC3 dots per cell was determined with a fluorescence microscopy for at least 100 GFP-LC3-positive cells.CCCAAGCTTATGCTGTGCTTCCTGAGGGGAATG (forward) and CCGCTCGAGTCAAATATCACCATTGATTTCTGTG (reverse), and subsequently digested with BamH I and Xho I and cloned in to the BamH I and Xho I sites of pcDNA4/HisA . pEGFP-N1-DRAM1 was generated through PCR primer: ATAGAATTCATGCTGTGCTTCCTGAGGGGA (forward) and CCGGGATCCTAATATCACCATTGATTTCTGTG(reverse), and products were T-A cloned in pMDTM19-T Vectors  and digested with EcoR I and BamH I and cloned into pEGFP-N1 . Transfection of cells with expression plasmids was performed using Lipofectamine 2000 .PcDNA4-DRAM1-His was generated by PCR from the I.M.A.G.E. clone for DRAM1 (Clone ID: NM_018370) with: CCACGATGTATACAAGATA and (2) CCACAGAAATCAATGGTGA. Negative siRNA TAAGGCTATGAAGAGATAC, were synthesized by GenePharma . The siRNA oligos used to target DRAM1 genes were previously validated and described in the following articles Small interfering RNAs (siRNA) targeting the following mRNA: TP53, AAGACUCCAGUGGUAAUCUAC; DRAM1, (1) For immunofluorescence microscopic examination, cells were plated on 12-mm Poly-L-Lysine-coated cover slips and cultured for 24 h, then cells were treated with siRNA and drugs. Cells were washed in PBS, fixed with 4% paraformaldehyde in PBS at 4°C for 10 min, and then washed again with PBS. The cells were permeabilized with 0.25% Triton X-100, and were then blocked with 10% normal goat serum (NGS) for 15 min. Primary antibodies: a rabbit polyclonal antibody against LC-3 , a goat polyclonal antibody against cathepsin D  and a rabbit polyclonal antibody against LAMP2  diluted in PBS were added to the cells and left for overnight at 4°C. The cover slips were washed three times before incubation with secondary antibodies using the same procedure as for the primary antibodies. The cover slips were mounted on slides with mounting medium  and were examined with a laser scanning confocal microscopy .The pattern of distribution of exogenously expressed GFP-LC3 in A549 cells was observed with fluorescent microscopy. GFP-LC3 dot formation was quantified by counting 500 GFP-LC3-positive cells and expressed as the ratio of the number of cells with at least 5 GFP-LC3 dots and the number of GFP-LC3-positive cells. The assays were independently performed by two investigators in a blinded manner and similar results were obtained.Western blot analysis was performed as scribed previously 2. Lysosomes were loaded overnight with 70000 MW FITC-dextran . and 0.5 mg/mL dextran-coupled Oregon Green 488  in DMEM supplemented with 10% FBS, chased for 2 h at 37°C with 5% CO2 in DMEM (10% FBS) to allow complete transfer of dextrans to lysosomes, and washed to remove residual dextran. Non-attached cells were removed by rinsing with PBS and the cover slips were immediately placed in a cuvette filled with growth medium or PBS and pH was estimated from excitation ratio measurements as described previously For lysosomal pH estimation, A549 and Hela cells were seeded on circular glass cover slips and grown to conﬂuence in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum  at 37°C, 5% CO8 cells in 2 ml) in homogenization buffer  and homogenized in a tight-fitting glass Dounce homogenizer. The homogenate was centrifuged  to remove unbroken cells and the nuclei. The supernatant was centrifuged  to remove the large organelle such as mitochondrial. The supernatant was centrifuged  to obtain the light organelle including lysosomes. The precipitation layered over 10 ml of a 27% Percoll  solution in homogenization buffer, underlayered with 0.5 ml of a 2.5 M sucrose solution. Centrifugation was done in a SW41Ti rotor  for 1.5 h at 35000 g. The layer of crude lysosomes of about 1.5 ml was collected at the bottom and then was centrifuged  to remove the other light organelle including mitochondrial at the bottom of the tube. Lysosomal fractions were equilibrated for up to 1 h in 125 mM KCl, 1 mM EDTA, and 20 mM Hepes (pH 7.5). Fluorescence was recorded continuously with excitation at 490 nm and emission at 520 nm. Upon addition of ATP , a progressive decrease in fluorescence intensity was observed, indicative of intralysosomal acidification + salt, pH 7.5, 5 mM), bafilomycin A1 (1 µM).FITC-dextran loaded A549 and Hela cells were prepared as described above. After washing in PBS, cells were resuspended  followed by Dunnett t-test or multiple means comparisons by Tukey’s test. Differences were considered significant when p<0.05.The present study examined if autophagic and apoptotic pathways are activated in A549 cells after 3-NP treatment. The results showed that 3-NP-induced a significant increase in the protein levels of DRAM1 from 3 to 72 h, with a peak induction at 24 h after 3-NP treatment . The speIt was reported that DRAM1 is a TP53 target gene. We determined the TP53 dependency in 3-NP-induced DRAM1 expression. In H1299 cells which lack of TP53, 3-NP only slightly induced DRAM1 expression, while in A549 cells which express wt TP53, 3-NP robustly induced the expression of DRAM1 . The simA). In addition, 3-NP-induced reduction of SQSTM1 was blocked by DRAM1 siRNA in A549 cells  and chloroquine (20 µM) were used to evaluate if inhibition of lysosomal functions produces effects similar to knock-down of DRAM1. Many autophagy inhibitors act on post-sequestration steps and agents, such as bafilomycin A1, that blocks autophagy activity are known to cause accumulation of autophagosomes 3-NP acts as an irreversible inhibitor of succinate dehydrogenase and thus results in an impairement of energy metabolism, oxidative stress and activation of glutamate receptors To investigate the underlying mechanism by which DRAM1 regulates autophagy, we investigated the effects of DRAM1 on autophagosome clearance. Colocalization of EGFP-DRAM1 and LysoTracker fluorescence or DRAM1 and LAMP2 immunoflurescence confirmed predominant lysosomal localization of expressed DRAM1. We first tested if DRAM1 has an effect on autophagosome turnover following induction with rapamycin. Rapamycin can stimulate the formation of autophagosome through inhibiting mTOR. Upon removal of rapamycin, autophagosomes should be cleared if autophagy pathway is normal. The present study demonstrated that rapamycin increased the abundance of autophagosomes and the number of autophagosomes returned towards the basal levels 6 h after withdrawal of rapamycin. Knock-down of DRAM1 reduced the rate of clearance of autophagosomes after rapamycin withdrawal. Galavotti et al reported that knock-down of DRAM1 inhibited targeting of SQSTM1 to autophagosomes and reduced its degradation The abundance of autophagosomes is balanced by the formation and clearance of autophagosomes. After the formation, the turn-over of autophagosomes is largely determined by the process of fusion between autophagososmes and lysosomes and degradation of autophagy contents by lysosomal enzymes. mRFP-GFP tandem fluorescent-tagged LC3 showed both GFP and mRFP signal of LC3 before the fusion with lysosomes, and exhibited only the mRFP signal when LC3 transmit into lysosomes because of lysosomal acidic environment and degradation +ATPases or VATPases. Proton transport into intracellular organelles is primarily mediated by ATPdependent proton pumps. These pumps are therefore central to pH homeostasis in organelles. Autophagosomes and their contents are cleared upon fusing with late endosomes or lysosomes containing cathepsins, other acid hydrolases, and vacuolar [H+] ATPase(v-ATPase) Lysosomes are rich in hydrolytic enzymes and are responsible for the degradation of intracellular materials captured by autophagy Lysosomes play important roles in autophagy. To test if the effects of DRAM1 on lysosomal functions are responsible for DRAM1-mediated autophagy activation after 3-NP treatment, the present study assessed the effects of lysosomal inhibitors on autophagosome accumulation in the presence of 3-NP. The results showed that elevating lysosomal pH and inhibiting lysosomal enzymes both increased accumulation of autophagosomes and inhibited cathepsin D activation. These results largely replicated the effects of knock-down of DRAM1 and suggested that DRAM1 probably regulated autophagy flux through lysosomes.It should be pointed out that DRAM1 appears regulate autophagy in both early and later stages of autophagy. DRAM1 can increase the formation of autophagosomes and the clearance of autophagosomes. These effects may work through the same mechanism as DRAM1 is a lysosomal protein and may regulates dynamics of lysosomal membranes to increase V-ATPase activity and to facilitate membrane recycle for autophagosomal formation.In conclusion, current data indicate that DRAM1 regulates autophagosome clearance through promoting lysosomal acidification and activation of lysosomal enzymes. The fusion of autophagosomes with lysosomes is an important step for autophagic degradation. In order to fully understand the role of DRAM1 in autophagy flux, the effects of DRAM1 on the fusion process between autophagosomes and lysosomes needs to be studied in the future.Figure S1DRAM1 mediated autophagy activation and lysosomal acidification in Hela cells. (A) Hela cells were transfected with DRAM1 siRNA or a non-silencing siRNA. Left: Forty-eight h after transfection of cells with DRAM1 siRNA, cells were harvested and protein levels of DRAM1 and LC3 were analyzed with immunoblotting. Right: Twenty-four hours after transfection of cells with DRAM1 siRNA, cells were treated with 3-NP (500 µM). Cells were harvested and protein levels of DRAM1 and LC3 were analyzed with immunoblotting 24 h after 3-NP. Densities of protein bands were analyzed with Sigma Scan Pro 5 and normalized to the loading control (β-actin). The data are expressed as percentage of control (non-silencing siRNA group). Bars represent mean±SE; n = 4. Statistical comparisons were carried out by ANOVA followed by Dunnett t-test. **P<0.01 (DRAM1 siRNA group vs. non-silencing siRNA group). ##P<0.01 (3-NP treated group vs. control group). $$P<0.01 (DRAM1 siRNA group vs. non-silencing siRNA group with 3-NP treatment). (B) Representative images of GFP-LC3 fluorescence in Hela cells transfected with GFP-LC3 and treated with DRAM1 siRNAs in the presence or absence of 3-NP (500 µM). Number of cells with GFP-LC3 dots was scored in 100 GFP-LC3-positive cells. N: the nucleus. Thin arrows: GFP-LC3 dots. The scale bar represents 10 µm Bars represent mean±SE; n = 4. Statistical comparisons were carried out by ANOVA followed by Dunnett t-test. **P<0.01 (siRNA group vs. non-silencing siRNA group). (C) Lysosomal pH was measured ratio-metrically using fluorescent dextrans in Hela cells. WT Hela cells and DRAM1 siRNA1-treated cells were loaded with the pH-sensitive fluorescent dextrans by endocytosis for 1 h at 37°C and then subjected to pulse-chase assay in the presence or absence of the 3-NP (500 µM). Lanes 2 and 4 depict pH values obtained with FITC-dextran after the addition of 500 nM 3-NP. The data are expressed as percentage of control (non-silencing siRNA cells). Bars represent mean±SE; n = 4. Statistical comparisons were carried out by ANOVA followed by Dunnett t-test. **P<0.01 (DRAM1 siRNA group vs. non-silencing siRNA group). ##P<0.01 (DRAM1 siRNA group vs. non-silencing siRNA group with 3-NP treatment). (D) Lysosomal V-ATPase activity was inhibited in DRAM1 siRNA1-treated Hela cells. Lysosomes from control cells and DRAM1 siRNA1-treated cells were loaded with FITC-dextran . Hela cells were then homogenized and used for in vitro-acidification assays. Fluorescence was recorded continuously with excitation at 490 nm and emission at 520 nm. Upon addition of ATP, a progressive decrease in fluorescence intensity was observed, indicative of intralysosomal acidification. The decrement was reversed by bafilomycin A1, a V-ATPase inhibitor.(TIF)Click here for additional data file.Figure S2Activity of DRAM1 antibody was blocked by DRAM1 peptide. (A) Cells were harvested and immunoblot analysis of DRAM1 protein levels in A549 and Hela cells. Left: No peptide incubated with DRAM1 antibody before primary antibody incubation. Right: DRAM1 peptide was incubated with DRAM1 antibody for 30 min at 37°C before primary antibody incubation. (B) Cells were processed for immunofluorescence using DRAM1 antibodies (green) and DAPI  in A549 and Hela cells, and was assessed with a confocal microscopy. Left: No peptide incubated with DRAM1 antibody before primary antibody incubation. Right: DRAM1 peptide was incubated with DRAM1 antibody for 30 min at 37°C before primary antibody incubation. N: the nucleus. Thin arrows: anti-DRAM1 fluorescence.(TIF)Click here for additional data file.


Geobacter metallireducens GS-15, has been determined at 2.6 Å resolution using single-wavelength anomalous dispersion method. The structure contains a two-stranded anti-parallel β-sheet that are surrounded by two helices on each face, and reveals a Zn ion bound in each monomer, coordinated by residues Cys38, Cys41, Cys73, and Cys76, which likely plays an important structural role in stabilizing the overall conformation. Structural homologs of Gmet_0936 include Hpy99I endonuclease, phage T4 endonuclease VII, and other HNH endonucleases, with these enzymes sharing 15–20% amino acid sequence identity. An overlay of Gmet_0936 and Hpy99I structures shows that most of the secondary structure elements, catalytic residues as well as the zinc binding site (zinc ribbon) are conserved. However, Gmet_0936 lacks the N-terminal domain of Hpy99I, which mediates DNA binding as well as dimerization. Purified Gmet_0936 forms dimers in solution and a dimer of the protein is observed in the crystal, but with a different mode of dimerization as compared to Hpy99I. Gmet_0936 and its N77H variant show a weak DNA binding activity in a DNA mobility shift assay and a weak Mn2+-dependent nicking activity on supercoiled plasmids in low pH buffers. The preferred substrate appears to be acid and heat-treated DNA with AP sites, suggesting Gmet_0936 may be a DNA repair enzyme.The crystal structure of a putative HNH endonuclease, Gmet_0936 protein from  Geobacter bacteria grow under anaerobic conditions in soils and aquatic sediment. Geobacter species can oxidize organic compounds and metals, including iron and manganese in anaerobic environment leading to the production of electrons which can be harnessed in bioreactors Geobeacter species in a microbial electrolysis cell Geobacter’s metabolic activities. Thus, Geobacter bacteria are important target for study in both bio-remediation applications and development of renewable energy. Geobacter metallireducens GS-15 was first isolated from aquatic sediment G. metallireducens GS-15 harbors restriction-modification (R-M) systems, presumably to fight off phage infections and invasion of foreign DNA. There are predicted restriction-modification (R-M) systems including a type IIS  and a type III , an orphan C5 methylase and an Mrr-like type IV restriction enzyme (see REBASE) 2. Companion DNA methyltransferase (MTase) is not found adjacent to Gmet_0936 so it is unlikely that this putative HNH endonuclease is part of an active R-M system. Gme_0936, however, is located in proximity to Gmet_0935, a predicted RNase HI homolog, and to Gmet_0939, a gene predicted to be involved in repair of stalled DNA replication fork.The HNH endonuclease catalytic domain with a zinc ribbon (ββα-Me fold) has been found in type II restriction endonucleases (e.g. Hpy99I and PacI), non-specific endonucleases (Colicins), DNA repair enzymes (MutS), and homing endonucleases (e.g. I-HmuI). Therefore, it is important to understand the structure and biochemistry of this group of enzymes. The goal of this work is to solve the crystal structure of Gmet_0936 protein and to study the enzymatic activity of this putative endonuclease. Here we report the expression, purification, and structural determination of Gmet_0936 at 2.6 Å resolution. In addition, we report a weak DNA binding activity of Gmet_0936 protein and the DNA nicking activity of the wt enzyme and its variant N77H. The possible biological function of Gmet_0936 (Gme HNH endonuclease or Gme HNHE) is discussed.Geobacter metallireducens (Gme) HNH endonuclease was cloned, expressed, and purified following standard protocols developed by the Northeast Structural Genomics Consortium (NESG) for production of selenomethionine enriched protein sample 6, H6 denoting a 6xHis-tag) that facilitate protein purification. E. coli BL21 (DE3) pMGK cells, a rare codon translation enhanced strain, were transformed with pGmR87-21.1, and cultured in MJ9 minimal medium supplemented with selenomethionine, lysine, phenylalanine, threonine, isoleucine, leucine, and valine for the production of selenomethionine-labeled HNHE The full-length gene Gmet_0936 of Crystals of Gme HNHE were obtained by the hanging-drop vapor diffusion method. One to three µl of concentrated protein solution  sodium azide and 5 mM dithiothreitol) were mixed with 1 µl of the reservoir solution consisting of 0.2 M magnesium sulfate, 20% (v/v) polyethylene glycol 8000, and 100 mM Tris-HCl (pH 8.5), followed by incubation at 4°C for 1 week. The crystals were cryo-protected using paratone-N and flash-frozen in liquid propane for data collection.free of 0.266 and standard R factor of 0.206  X-ray data were collected at wavelength 0.979 Å from a single crystal of the Gmet_0936 protein at 100 K on beam line X4A at the National Synchrotron Light Source at Brookhaven National Laboratory. The diffraction images were processed with the program package HKL2000 of 0.206 . The RamE. coli codons was subcloned into a T7 expression vector pET21b (the insert was flanked by NdeI and XhoI sites). The C-terminal 6xHis-tagged protein, Gmet_0936 or its mutant variants were partially purified through a nickel spin column from 10–20 ml IPTG-induced cell culture. For medium-scale purification, 1 to 2 L of cells ER2566 [pET21b-gmet_0936] were grown at 37°C to late log phase and IPTG was added to a final concentration of 0.5 mM for protein induction at 30°C for 3 h. Gmet_0936 (H6) protein was purified through a nickel column by gravity flow using a protocol recommended by the manufacturer . It was further purified by chromatography through a HiTrap Heparin HP column . Gmet_0936 (H6) protein peak eluted at the NaCl concentration of 0.5 to 0.55 M. Alternatively, the NdeI-XhoI fragment was inserted into pTYB1 vector to be in fusion with intein and chitin-binding domain (CBD). The fusion protein Gmet_0936-intein-CBD was purified from a chitin column (NEB) and Gmet_0936 was cleaved off by DTT and eluted. Gmet_0936 was further purified by chromatography through a HiTrap Heparin HP column , protein was eluted by a NaCl gradient of 50 mM to 1 M and eluted fractions were analyzed by SDS-PAGE. Purified fractions containing Gmet_0936 protein was stored in 0.25 M NaCl, 20 mM Tris-HCl, pH 7.5, 50% glycerol, 1 mM DTT at −20°C. Reaction buffer contains 50 mM NaCl, 10 mM Tris-HCl, pH 6 to 8, supplemented with divalent cations  as specified in each reaction. Digestion was carried out at 37°C for 1–2 h or overnight, and reaction was stopped by addition of a stop dye solution with EDTA (10 mM) and SDS (0.1%).A synthetic gene coding for Gmet_0936 in pIDTSMART-Kan (IDT) with optimized FAM-labeled duplex oligos were incubated with 6xHis-tagged N77H or Gme HNHE at room temperature for 20 min and the DNA-protein complexes were resolved in a 10% PAG gel. The image of DNA-protein complex was derived by scanning on a variable mode imager .G ATA GTT ACC GGA TAA GGC GCA GCG G 3′ (ACCGG-top).Oligo 1 sequence: 5′ AC CTG CGC CTT ATC CG G TAA CTA TCG T-FAM 3′ (ACCGG-bottom).Oligo 2 sequence: 5′ CCGThe published procedure for acid and heat damaged (depurinated) DNA was followed E. coli competent cells ER2566 and transformants were selected on LB agar Amp plates. The desired mutations were confirmed by DNA sequencing of the alleles on plasmids.Gmet_0936 variants were created by PCR mutagenesis using a pair of non-overlapping primers and Phusion DNA polymerase® (NEB). PCR products were purified through spin columns, phosphorylated by T4 polynucleotide kinase and ligated by T4 DNA ligase. Ligated DNA was used to transform The PDB entry codes (coordinates and structure factors) have been deposited in pdb database and received the accession number: 4H9D.Geobacter species and some members of Geobacteraceae, Desulfuromonadales, and Pelobacteraceae. For example, Gmet_0936 shares 75% aa sequence identity to a putative HNHE (protein ID: YP_001231596) from Geobacter uraniireducens. It also shares 76% aa sequence identity to a putative HNHE (protein ID: NP_953119) encoded in the genome of Geobacter sulfurreducens PCA strain. The presence of a large number of close homologs suggests that Gmet_0936 and its relatives may have some unknown biological function, possibly in DNA repair or recombination.A BlastP search for homologous proteins in GenBank using Gmet_0936 as a query identified over 50 small putative HNH endonucleases (92 to 133 aa in length) with more than 50% amino acid (aa) sequence identity. The top ten close homologs with the predicted secondary structure are shown in G. metallireducens GS-15 was determined at 2.6Å resolution, using selenomethionyl (Se-Met) derivatized protein and single-wavelength anomalous dispersion method of phase determination. The structure model was refined to working and free R-factors of 0.206 and 0.266, respectively. The data processing and refinement statistics are presented in 3. The conformations of the three molecules in the asymmetric unit are similar, with rms distance of ∼0.7 Å among their equivalent Cα atoms. The structure revealed a Zn ion associated with each monomer, coordinated by residues Cys38, Cys41 (in the long loop connecting helix α2 and strand β1), Cys73, and Cys76   Fig. 2A.Vibrio vulnificus (1OUO) Vibrio salmonicida (2PU3) Z scores of 4.3, 3.9, 3.5, and 3.5, respectively. The root mean squared distance for ∼65 equivalent Cα atoms is 3.1–3.6 Å, and the proteins share 15–20% amino acid sequence identity with Gme HNHE. An overlay of the structures of Gme HNHE and Hpy99I is shown in E. coli, and the lack of apparent DNA digestion pattern in Mg++ buffer led to the conclusion that Gme HNHE is not a typical type II restriction enzyme (see below for DNA nicking activity).The Gme endonuclease belongs to the family of HNH endonucleases which is also known as ββα-Me endonucleases. This conserved ββα Me fold with a bound zinc ion has been found in a wide range of nucleic acid enzymes including non-specific endonucleases, type II restriction enzymes, homing endonucleases, holiday junction resolvase, and DNA repair enzymes. We next performed homolog search of known structures in pdb. Structural homologs of the Gme HNHE was identified with the program DaliLite 42-R93-Y100-N113, further illustrating the diversity of the HNH family enzymes Sequence analysis suggests that residues Asp53, His54, Asn68 and Asn77 are in the active site of the HNH endonuclease. Residues Asp53 and His54 are at the end of strand β1, on the surface of the monomer, and Asn68 and Asn77 are located close to these two residues Fig. 2A.E. coli McrA also have the conserved HNH catalytic domain and a zinc binding site. Sco_McrA cleaves phosphorothioated DNA as well as methylated DNA and prefers Mn2+ or Co2+ for catalytic activity 2+ –dependent catalytic activity is shared by Gme HNHE, Sco_McrA endonuclease, and some phage/prophage encoded nicking enzymes. E. coli McrA restricted incoming 5 mC modified λ phage or plasmid DNA or 5 hmC-modified T4gt Studies in solution indicate that the Gme HNHE is primarily a dimer (see molecular sizing chromatography results below). Two of the three molecules in the crystallographic asymmetric unit form a dimer Fig. 3A,2+, Co2+, or Ni2+) with binding affinity for Zn2+ three order of magnitude higher than other divalent cations. The metal ion is coordinated by three His residues and Zn2+ is not required for the endonuclease activity, but necessary for structure stabilization The Zn binding site of Gme HNHE is reminiscent of the zinc ribbon of the 134-aa nuclease domain of Colicin E9 containing a zinc-finger-like HNH motif that binds divalent transition metal ions . By multiple sequence alignment with other known HNH endonucleases and by structure-guided analysis, the possible catalytic residues of Gmet_0936 protein are D53, H54, N68, and N77. N77 position is usually occupied by a His residue in many HNH endonucleases  6) was partially purified  and assayed for endonuclease activity.The 6xHis-tagged (13.3 kDa) and none-His-tagged Gmet_0936 protein (12.7 kDa) purified from chitin/Heparin columns showed slightly larger apparent molecular size than the predicted molecular masses (6) and its N77H (H6) variant is still unknown, the bound complexes are probably resulted from non-specific DNA binding activity. In the control experiment, the Bsu HNH endonuclease which has a DNA nicking specificity of CG↓GT (ACCG) bound the duplex oligos and formed a high molecular weight complex (6) and N77H (H6) variant are weak DNA binding proteins. Gme HNHE may need other DNA binding partner(s) for high affinity DNA binding.6) enzymes in buffers with different divalent cations such as Mg2+, Mn2+, Zn2+, Ca2+, Co2+, or Ni2+ at various pH levels (pH 6 to 8). N77H (H6) enzyme displays detectable DNA nicking activity in Mn2+ buffers at low pH (pH 6 to 7) (data not shown). Purified Gme HNHE and N77H (H6) enzymes display weak DNA nicking activity in overnight digestions at pH 7 in a Mn++ buffer. 6) and Gme HNHE enzymes nick dsDNA (φX174 and M13 RFI DNA) and they also changed the migration pattern of ssDNA  in the overnight digestion. Both enzymes do not appear to cleave pre-nicked φX174 RFII DNA (lanes 4–6). Multiple attempts to determine the nicking specificity (nicking site) of N77H (H6) were not successful when nicked and gel-purified pBR322 DNA was used as templates for run-off sequencing, suggesting that N77H (H6) endonuclease nicks plasmid DNA non-specifically. In a control experiment, nicked pBR322 by site-specific and strand-specific nicking enzymes (N.φGamma) gave rise to sharp drop in sequencing peaks in run-off sequencing (data not shown).We next digested various DNA substrates with purified Gme HNHE and N77H (H6) endonuclease is more active in low pH buffers  it was suspected that the rate of DNA depurination was enhanced at the lower pH levels (pH 6–7), resulting in the apparent elevated DNA nicking activity. Therefore we tested the nicking activity of Gme N77H (H6) enzyme on acid and heat damaged pBR322 DNA. Fig. S2A shows the partial nicking activity of N77H (H6) on regular pBR322 in an Mn++ buffer at pH 7.0. Fig. S2B shows that following acid and heat treatment for 2 h, some supercoiled DNA was converted to nicked circular DNA (breaks of DNA backbones in addition to depurination) . Most of the supercoiled DNA was converted to nicked circular DNA after 90 min digestion by N77H (H6). It was concluded that the DNA nicking activity of N77H (H6) could be enhanced by acid and heat treatment of plasmid DNA. However, we cannot exclude other catalytic activity for this enzyme, which may be more physiologically relevant.Since Gme N77H . In addition, K14E/N77A transformation into a T7 expression strain (ER2566) was only about 0.4% of the wt Gme HNHE (data not shown), indicating that K14E/N77A may be toxic to the expression host, possibly due to its tight DNA binding and interference with transcription or DNA replication . The Gme H54A variant is also toxic to E. coli with reduced transformation efficiency. The partially purified mutant proteins D53A, H54A, N68A displayed reduced nicking activity on pBR322  (data not shown). The activity of K14E/N77A cannot be determined due to the poor expression and lack of purified protein. From the mutagenesis result, it was concluded that H54, N68, and N77 are critical for both enzyme activity and for maintaining proper structural folding as well. The role of D53 residue is less important for structure integrity and it’s negative charge of side chain may contribute to the catalytic site by stabilizing a metal ion.We also constructed four other 6xHis-tagged variants: D53A, H54A, N68A, and N77A; and these four mutant proteins were partially purified from nickel spin columns. The protein expression level in crude cell lysate and in nickel column purified fractions was normal for the D53A variant, but much reduced for the H54A and N68A variants (∼5 to 10% of wt), and poorly expressed for the K14E/N77A double mutant (we were unable to isolate N77A single mutant since Asn77 to Ala substitution was accompanied by a secondary mutation K14E) (6) variant in nicking pUC19 DNA and a pUC-derivative that contains a long stretch of [AT]20 tracks that can form a cruciform structure. Both plasmids were nicked non-specifically by N77H (H6) endonuclease, with equal nicking activity on the plasmid with a cruciform structure (data not shown). We concluded that Gme HNHE is unlikely a holiday junction resolvase by itself.Due to the structure similarity in the HNH catalytic domain of Gme HNHE and phage T4 endonuclease VII, we tested N77H (Hav) of Gmet_0936 protein was determined by Kav = (ve−vo)/(vt−vo) where ve is the elution volume, vo is the void volume of the column and vt is the total column volume. vo was determined empirically from the elution time of blue dextran and vt was the total column volume 3 ml. A standard curve was created by obtaining the Kav values of the standard protein ribonuclease A (13.5 kDa), ovalbumin (43 kDa) and conalbumin (75 kDa)  run under the same conditions. The Kav values for two enzyme concentrations and the standard proteins are represented by open circles and block squares, respectively. The Kav values and the derived apparent molecular weight of Gmet_0936 protein are 0.899 and 0.891, corresponding to 26.2 kDa and 25.3 kDa for the 4 mg/ml and 8 mg/ml injection, respectively . The apparent molecular weight of the Gme HNHE is consistent with a dimer configuration.To determine the oligomerization state of Gme HNHE, we performed molecular sizing column chromatography for the enzyme (purified by chitin and Heparin columns). Ten µl of the purified non-His-tagged Gme HNHE (4 or 8 mg/ml) was injected into a Superdex 200 5/150 GL column . The partition coefficient , Gme HNHE (H6) was purified to near homogeneity from a nickel column and a gel filtration column. Thawed aliquot of the protein crystallization stock was subjected to analytical gel filtration on a Shodex 802.5 column  running at 4°C in 100 mM NaCl, 0.025% (w/v) NaN3, 100 mM Tris-HCl (pH 7.5). The column eluant was monitored using static light scattering and refractive index detectors . Debye analysis shows a molecular mass of 23.9 kDa, indicating that Gme HNHE (H6) is a dimer (data not shown), which is consistent to the result derived from molecular sizing column described above for the non-His tagged protein. In term of cleavage/nicking mechanism, it is not clear why Gme HNHE only nicks DNA and does not cause extensive ds breaks on acid/heat damaged DNA.To analyze the oligomerization state of the Gme HNHE with a C-terminal 6xHis tag  variant shows improved non-specific DNA nicking activity on acid and heat damaged plasmid DNA. Therefore, we suspect that Gme HNHE may play a role in DNA repair and/or recombination. The structural similarity of Gme HNHE and the C-terminus of Hpy99I restriction enzyme suggested divergent evolution of the HNH (HNN/HNK) family endonucleases: one towards possible DNA repair funtion (nicking of damaged DNA for homologous recombination repair) and the other towards DNA restriction by fusion with a sequence-specific DNA recognition domain. The structure of Gme HNHE reported here complements the structural information derived from other HNH family nucleases including His-Cys box homing endonuclease, restriction endonuclease, DNA repair enzyme, holiday junction resolvase, and non-specific Colicin endonucleases.The biological function of Gme HNHE is unknown. Due to its weak DNA binding activity, Gme HNHE may require other protein partners to be recruited to DNA lesion sites, and introduce nicks at or near AP sites. With the availability of the 6xHis-tagged Gme HNHE and N77H variant, it is now possible to identify the protein interaction partner from cell extracts of Bacillus subtilis (hlpB or yisB gene) B. subtilis and possibly has similar function in other bacteria. The HlpB (100 aa residues) and Gme HNHE (104 aa residues) only share 11% aa sequence identity. Based on the biochemical properties of HlpB and Gme HNHE, we suspect that Gme HNHE likely plays a different DNA repair function from that of HlpB protein. More genetic and biochemical studies are needed to pinpoint the biological function of Gme HNHE.Recently, a protein called HlpB essential for cell viability with the conserved HNH motif was discovered in Geobacter metallireducens GS-15 was determined at 2.6 Å resolution. The structure contains a two-stranded anti-parallel β-sheet surrounded by two helices on each face, and reveals a Zn binding site in each monomer, coordinated by residues Cys38, Cys41, Cys73, and Cys76. Structural homologs of Gmet_0936 include Hpy99I endonuclease, phage T4 endonuclease VII, and other HNH endonucleases with the conserved ββα-Me structural fold. An overlay of Gmet_0936 and Hpy99I structures shows that most of the secondary structure elements, catalytic residues as well as the zinc binding site (zinc ribbon) are conserved. However, Gmet_0936 lacks the N-terminal domain of Hpy99I, which mediates DNA binding as well as dimerization. Purified Gmet_0936 forms dimers in solution and a dimer of the protein is observed in the crystal, but with a different mode of dimerization as compared to Hpy99I. Gmet_0936 and its N77H variant show a weak DNA binding activity in a DNA mobility shift assay and a weak Mn2+-dependent nicking activity on supercoiled plasmids in low pH buffers. The preferred substrate is acid and heat-treated DNA with AP sites, suggesting that Gmet_0936 may be a DNA repair enzyme. Site-directed mutagenesis indicated that the catalytic residues His53, N68, and N77 are also important for maintaining proper structure folding of the protein.The crystal structure of Gmet_0936 protein from Figure S1SDS-PAGE analysis of purified His-tagged and non-His-tagged proteins. A. Partially purified proteins: 6xHis-tagged Gme HNHE with the non-native C-terminal amino acid (aa) residues LEH6, predicted molecular mass: 13,264 Dalton, pI = 6.71. Gme HNHE with non-native C-terminal residues LEGSS following intein cleavage, predicted molecular mass: 12,672 Dalton, pI = 5.43. Lane 1, IPTG-induced and clarified cell lysate ; lanes 2 and 3, flow-through (FT) from the nickel column; lanes 4 and 5, eluted fractions containing Gme HNHE (H6) protein; lanes 6 and 8, eluted fractions containing Gme HNHE from a Heparin column purification; lane 7, chitin column eluent containing Gme HNHE after DTT cleavage; lane 9, protein ladder (NEB). B. SDS-PAGE analysis of partially purified N77H (H6) protein. Lane 1, eluent from a nickel column; lane 2, IPTG-induced and clarified cell lysate ; lane 3, total proteins in cell lysate; lane 4, protein ladder. C. SDS-PAGE analysis of purified N77H (H6) protein eluted fractions from a Heparin column.(TIFF)Click here for additional data file.Figure S26) DNA nicking activity assays on regular pBR322 and acid/heat damaged pBR322 DNA. A.N77H (H A time course of N77H (H6) nicking activity on pBR322. PstI, linear DNA; Nt, nicked circular form by Nt.BspQI; nicking reaction time course (10 to 90 min) at 37oC in an Mn++ buffer (1 mM). zero = DNA plus buffer, no enzyme added. B. A time course of N77H (H6) nicking activity on acid/heat damaged pBR322 . Following acid/heat treatment, a fraction of the supercoiled DNA had been converted to linear and nicked circular forms (see zero time point).(TIFF)Click here for additional data file.Figure S3SDS-PAGE analysis of Gme HNHE variants in cell extract and in partially purified protein fractions from nickel spin columns. A. SDS-PAGE analysis of WT Gme HNHE and its variants. Lane 1–2, protein ladder and purified Gme HNHE; lanes 3–9, IPTG-induced cell extracts containing WT Gme HNHE, and its variants D53A, H54A, N68A, N77H, and K14E/N77A (two isolates). B. SDS-PAGE analysis of partially purified WT Gme HNHE and its variants. Lane 10–11, protein ladder and purified WT Gme HNHE (Heparin); lanes 12–18, partially purified enzymes from nickel spin columns: WT Gme HNHE and its variants D53A, H54A, N68A, N77H, and K14E/N77A (two isolates).(TIFF)Click here for additional data file.Figure S4Molecular sizing column chromatography to determine the oligomerization state of the Gme HNHE. The partition coefficient (Kav) of Gme HNHE was determined by Kav = (ve−vo)/(vt−vo) where ve is the elution volume, vo is the void volume of the column and vt is the total column volume. vo was determined empirically from the elution time of blue dextran and vt was the total column volume 3 ml. A standard curve was created by obtaining the Kav values of the standard protein ribonuclease A (13.5 kDa), ovalbumin (43 kDa) and conalbumin (75 kDa) run under the same conditions. The Kav values for two enzyme concentrations and the standard proteins are represented by open circles and block squares, respectively. The Kav values and the derived apparent molecular weight of Gme HNHE are 0.899 and 0.891, corresponding to 26.2 kDa and 25.3 kDa for the 4 mg/ml and 8 mg/ml injection, respectively.(TIFF)Click here for additional data file.


The results show that 48 h of FD induced a greater decrease in protein synthesis in type 2X and 2B fibers compared to type 1 and 2A fibers. Type 2X and 2B fibers also had the largest FD-induced decrease in total S6 protein and Ser240/244 S6 phosphorylation, respectively. Moreover, only type 2X and 2B fibers displayed a FD-induced decrease in cross-sectional area (CSA). Ten days of SA also induced fiber type-dependent responses, with type 2B fibers having the smallest SA-induced increases in protein synthesis, CSA and Ser240/244 S6 phosphorylation, but the largest increase in total S6 protein. Embryonic myosin heavy chain (MHCEmb) positive fibers were also found in SA muscles and the protein synthesis rates, levels of S6 Ser240/244 phosphorylation, and total S6 protein content, were 3.6-, 6.1- and 2.9-fold greater than that found in fibers from control muscles, respectively. Overall, these results reveal differential responses in the regulation of protein synthesis and fiber size between fiber types found within the same whole muscle. Moreover, these findings demonstrate that changes found at the whole muscle level do not necessarily reflect changes in individual fiber types.This study examined fiber type-dependent differences in the regulation of protein synthesis in individual muscle fibers found within the same whole muscle. Specifically, the  Mammalian skeletal muscles are heterogeneous tissues composed of different fiber types that are identified by their expression of specific myosin heavy chain (MHC) isoforms. For example, adult limb and trunk muscles are composed of four major fiber types; oxidative slow-twitch type 1 fibers, oxidative fast-twitch type 2A fibers, and glycolytic fast-twitch type 2X and 2B fibers, which express MHC isoforms 1, 2a, 2x and 2b, respectively. Over the last century, a large body of research has described the distinct genetic, structural, functional, metabolic and adaptive characteristics of these fiber types (for reviews see in vivo rates of protein synthesis at the single muscle fiber level. This method is based on the principles of the SUrface SEnsing of Translation (SUnSET) technique and it involves injecting animals with a low dose of puromycin  Recently, we developed a non-radioactive method for measuring Our finding of fiber type-dependent differences in basal rates of protein synthesis highlights the long standing question of whether physiological perturbations can alter protein synthesis in a fiber type-dependent manner ad libitum access to food and water unless otherwise stated. Before all surgical procedures, mice were anesthetized with an intraperitoneal (IP) injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). After tissue extractions, the mice were euthanized by cervical dislocation. All methods were approved by the Institutional Animal Care and Use Committee of the University of Wisconsin-Madison under the protocol #V01324.Male FVB/N mice, 8–10 wk old, were used for all conditions. Mice were housed under a 12 h light/dark cycle with Food deprived (FD) mice had food withdrawn for 48 h, with ad libitum access to water as previously described Mechanical overload was induced by synergist ablation (SA) surgery that involved the bilateral removal of the soleus and distal half of the gastrocnemius muscle, leaving the PLT as the sole plantar flexor muscle in vivo measurements of protein synthesis, a pair of mice (Ad lib and FD or Sham and SA) were anesthetized and then simultaneously given an IP injection of 0.04 μmol/g puromycin dissolved in 100 μl of phosphate buffered saline (PBS), as previously described 2-chilled isopentane.For r  =  0.9997, data not shown). The camera was mounted on a Nikon 80i epifluorescence microscope, and monochrome images were captured through TRITC, FITC and/or DAPI cubes and merged with Nikon NIS-Elements D software as previously described Emb). For MHCEmb fibers, the area and average puromycin signal intensity was expressed relative to the mean of sixty randomly selected, non-typed, fibers from the sham section on the same slide. All analyses were performed on images that had signal intensities within the linear range of the camera. The analyses were performed by investigators masked to the sample identity and involved the quantification of at least five independent pairs of muscles.Cross sections (10 μm thick) from the paired muscles were taken at the mid-belly and fixed in −20°C acetone for 10 min. Sections were warmed to room temperature for 5 min and then incubated in PBS for 15 min, followed by a 1 h incubation in solution A [PBS with 0.5% bovine serum albumin (BSA) and 0.5% Triton X-100] containing anti-mouse IgG Fab . After three 5 min washes with PBS, samples were incubated for 1 h with solution A containing primary antibodies  for 1 h. After three 5 min washes, samples were incubated for 1 h with solution A containing primary antibodies specific for MHC 1, 2a, 2x or 2b as described above, and either rabbit anti-ribosomal S6 protein antibody  or anti-rabbit phospho-S6 ribosomal protein (Ser240/244) XPcles see . For thet-tests, were used for all 2-group comparisons. One-way ANOVA with Newman-Keul post-hoc analysis was used for comparisons involving more than 2 groups. The statistical significance level was set at P<0.05.All statistical analyses were performed with GraphPad Prism 5.0 . Student's 2-tailed, unpaired To explore potential fiber type differences in the regulation of protein synthesis, we employed two experimental models that are known to induce a decrease [food deprivation (FD)] and an increase [synergist ablation (SA)] in skeletal muscle protein synthesis In the PLT muscle we identified four different fiber types: slow-twitch type 1 and fast-twitch types 2A, 2X and 2B  A–D. As As shown in 240/244 phosphorylation of the ribosomal S6 protein. This selection was based on previous studies which have shown that ribosomal S6 protein is phosphorylated at the Serine 240 and 244 residues by p70 ribosomal S6 kinase (p70S6K) S6K is a direct downstream target of the mammalian target of rapamycin complex 1 (mTORC1), and mTORC1 has been widely implicated in the regulation of translational efficiency 240/244 phosphorylation were used as a marker of potential mTORC1-mediated changes in translational efficiency.To gain insight into the mechanism(s) responsible for the observed fiber type-dependent changes in protein synthesis we performed IHC analysis for crude markers of translational capacity and translational efficiency. As a marker for translational capacity we measured the total amount of the ribosomal S6 protein , and that the magnitude of the increase in these fibers was similar to the increase in the total number of fibers Emb positive fibers. Therefore, we stained for the presence of fibers expressing MHCEmb and measured protein synthesis, Ser240/244 S6 phosphorylation and total S6 protein in these fibers when compared to a population of mixed fiber types randomly chosen from the adjacent Sham cross-section (Mixed Sham).Recently, we reported that SA induces a significant increase in the total number of muscle fibers per cross-section Emb positive fibers that were not present in Sham cross-sections. Consistent with the idea that these are newly formed fibers, the MHCEmb positive fibers were significantly smaller in CSA when compared to the Mixed Sham fibers  behind the fiber type-dependent response of protein synthesis to FD remains to be determined, however, recent studies at the whole muscle level may provide some insights. For example, FD induces an increase in the expression of the atrophy-related genes, myostatin and atrogin-1, with whole fast-twitch muscles showing greater increases in expression compared with whole slow-twitch muscles Another mechanism that could help to explain the greater FD-induced decrease in type 2X and 2B fiber CSA is fiber-type dependent differences in the expression of PGC1α. Recent evidence has shown that more oxidative fiber types have higher levels of PGC1α expression than glycolytic fiber types 240/244 phosphorylation, total S6 protein, protein synthesis and fiber CSA. These fiber type-dependent responses are likely due to a combination of the intrinsic fiber type-dependent differences in the molecular mechanisms that regulate protein synthesis and degradation, as well as, fiber type-dependent use patterns. Nevertheless, further work will be needed to define the exact molecular mechanism(s) responsible for these fiber-type dependent responses.In summary, relatively short-term FD induces fiber type-dependent changes in S6 Ser240/244 phosphorylation (2B ≈ 2X < 2A < 1) was slightly different from that observed with total S6. Specifically, the level of S6 Ser240/244 phosphorylation was not different between type 2X and 2B fibers. Furthermore, S6 Ser240/244 phosphorylation in type 1 fibers was higher than in type 2A fibers. Thus, it seems that, at least under basal conditions, rates of protein synthesis are not as closely associated with this marker of mTORC1 signaling, and presumably, translational efficiency. Nevertheless, our data clearly reveals fiber type-dependent differences in the basal levels of both total ribosomal S6 protein and S6 Ser240/244 phosphorylation and further work will be required to elucidate the molecular mechanisms responsible for these findings.In the process of investigating the effect of FD on translational capacity we noticed that sections from Ad Lib muscle had significant inter-fiber differences in the amount of the ribosomal S6 protein. This observation suggested that the basal expression of the ribosomal S6 protein may be fiber type-dependent. Indeed, our results showed that the total S6 protein signal intensity varied in the following order: 2B < 2X < 1 < 2A. These findings suggest that translational capacity may vary in a fiber type-dependent manner. This hypothesis is supported by the study of Habets et al 240/244 phosphorylation and total S6 protein were also observed in response to SA. Specifically, SA resulted in significantly larger increases in protein synthesis in type 1 and 2A fibers compared to type 2X and 2B fibers, with the increase in type 2X fibers being intermediate to 2A and 2B fibers. Moreover, this fiber type-dependent pattern of increases in protein synthesis was largely mirrored by a similar pattern of fiber type-dependent increases in fiber CSA. For example, type 2B fibers revealed the smallest SA-induced increase in both protein synthesis and CSA, while type 2A fibers had the largest increase in both these parameters. One potential mechanism behind these observations could be the result of differences in motor unit recruitment patterns Fiber type-dependent changes in protein synthesis, CSA, S6 Ser240/244 phosphorylation are not so congruent. For example, despite having the smallest SA-induced increase in protein synthesis, type 2B fibers had a 55–77% greater increase in total S6 protein compared to the other three fiber types. Furthermore, the SA-induced increase in S6 Ser240/244 phosphorylation was not different between type 2A and 2X fibers, and type 1 fibers had a larger increase in S6 Ser240/244 phosphorylation than type 2A fibers. These data suggest that the fiber type-specific regulation of protein synthesis and CSA in response to mechanical overload results from a complex combination of changes in translational efficiency and capacity. Interestingly, the SA-induced increase in Ser240/244 S6 phosphorylation, when expressed relative to the increase in total S6 protein, was 5.7-, 4.4-, 4.4- and 1.8-fold for type 1, 2A, 2X and 2B fibers, respectively. This suggests that, compared to type 1, 2A and 2X fibers, the increase in Ser240/244 S6 phosphorylation in type 2B fibers was largely due to an increase total S6 protein. This also suggests that, compared to type 1, 2A and 2X fibers, the increase in protein synthesis in type 2B fibers was driven to a greater extent by an increase in translational capacity than translational efficiency. Moreover, the fact that there was a relatively smaller increase in protein synthesis in type 2B fibers  and 48 h food deprived (FD) mice were frozen adjacent to one another, cross-sectioned, and then subjected to immunohistochemistry for rates of protein synthesis (puromycin) and different fiber types as described in A–H) Frequency histograms representing the effect of FD on the relative rate of protein synthesis (i.e. puromycin staining intensity) , and cross-sectional area , within a given fiber type. Inset values are presented as the mean ± SEM (n  = 60–250 fibers / group from 5 independent pairs of muscles). * Significant effect of FD, (P<0.05).(TIF)Click here for additional data file.Figure S2Synergist Ablation Induces Fiber Type-Dependent Changes in Protein Synthesis and Cross-Sectional Area. Plantaris muscles obtained from control (Sham) and 10 d synergist ablated (SA) mice were frozen adjacent to one another, cross-sectioned, and then subjected to immunohistochemistry for rates of protein synthesis (puromycin) and different fiber types as described in A–H) Frequency histograms representing the effect of SA on the relative rate of protein synthesis (i.e. puromycin staining intensity) , and cross-sectional area , within a given fiber type. Inset values are presented as the mean ± SEM (n  = 84–500 fibers / group from 6 independent pairs of muscles). * Significant effect of SA, (P<0.05).(TIF)Click here for additional data file.Figure S3240/244 Phosphorylated and Total Ribosomal S6 Protein.Fiber Type-Dependent Differences in Basal Ser Muscle sections from Ad Lib mice were subjected to immunohistochemistry for different fiber types and Ser240/244 phosphorylated S6 (P-S6 Ser240/244) or total S6, as described in A) P-S6 Ser240/244 and (B) total S6 in each fiber type was expressed relative to the mean value obtained in type 2A fibers from the same section. Values are means + SEM (n  = 59–300 fibers / group from 4–5 independent muscles). ∗ significantly different from all other fiber types, # significantly different from type 1 and 2A fibers (P>0.05).(TIF)Click here for additional data file.Figure S4240/244 Phosphorylated and Total Ribosomal S6 Protein.Food Deprivation Induces Fiber Type-Dependent Changes in Ser Plantaris muscles obtained from control (Ad Lib) and 48 h food deprived (FD) mice were frozen adjacent to one another, cross-sectioned, and then subjected to immunohistochemistry for different fiber types and Ser240/244 phosphorylated S6, or total S6, as described in A–H) Frequency histograms representing the effect of FD on the relative staining intensity of Ser240/244 phosphorylated S6 (P-S6 Ser240/244) , and total S6 , within a given fiber type. Inset values are presented as the mean ± SEM (n  = 60–250 fibers / group from 5 independent pairs of muscles). * Significant effect of FD, (P<0.05).(TIF)Click here for additional data file.Figure S5240/244 Phosphorylated and Total Ribosomal S6 Protein.Synergist Ablation Induces Fiber Type-Dependent Changes in Ser Plantaris muscles obtained from control (Sham) and 10 d synergist ablated (SA) mice were frozen adjacent to one another, cross-sectioned, and then subjected to immunohistochemistry for different fiber types and Ser240/244 phosphorylated S6, or total S6, as described in A–H) Frequency histograms representing the effect of SA on the relative staining intensity of Ser240/244 phosphorylated S6 (P-S6 Ser240/244) , and total S6 , within a given fiber type. Inset values are presented as the mean ± SEM (n  = 84–500 fibers / group from 6 independent pairs of muscles). * Significant effect of SA, (P<0.05).(TIF)Click here for additional data file.Figure S6240/244 Phosphorylated and Total Ribosomal S6 Protein in MHCEmb Positive Fibers.Cross-Sectional Area, Protein Synthesis, Ser Plantaris muscles obtained from control (Sham) and 10 d synergist ablated (SA) mice were frozen adjacent to one another, cross-sectioned, and then subjected to immunohistochemistry for MHCEmb and rates of protein synthesis (puromycin), Ser240/244 phosphorylated S6 (P-S6 Ser240/244), or total S6, as described in A) fiber cross-sectional area (CSA), (B) rate of protein synthesis, (C) amount of Ser240/244 phosphorylated S6 (P-S6 Ser240/244) and (D) total amount of S6 in MHCEmb positive fibers of SA muscles expressed relative to randomly selected fibers from sham muscles (Mixed Sham). Inset values are presented as the mean ± SEM (n  = 152–360 fibers / group from 6 independent pairs of muscles). ∗ Significantly different from mixed sham, (P<0.05).(TIF)Click here for additional data file.


Hepatitis C virus (HCV) is a major cause of chronic liver disease. However, little is known about the details of its assembly and secretion. Golgi-related proteins have been recently proven to have a key function in HCV secretion. Golgi protein 73 (GP73), a resident Golgi membrane protein, is a potential serum biomarker for the diagnosis of liver diseases and hepatocellular carcinoma. Previous studies have demonstrated the upregulation of GP73 in the liver samples and sera of HCV-infected patients. However, the function and regulatory mechanism of GP73 in HCV infection at the cellular level remain unknown. In this study, we examined the expression level of GP73 in HCV infected cells and its effect on HCV life cycle in cell culture systems. Both the protein expression and mRNA levels of GP73 significantly increased in HCV subgenomic replicon-harboring cells and HCV-infected cells, which imply that GP73 was upregulated by HCV infection. HCV production was significantly enhanced when GP73 was overexpressed, but dramatically inhibited when GP73 was silenced. However, the overexpression and knockdown of GP73 showed no evident effect on the entry, protein translation, RNA replication, and assembly of HCV, which indicates that GP73 enhanced the secretion process. Moreover, the coiled-coil domain of GP73 was required to increase HCV secretion. GP73 increased and interacted with apolipoprotein E, an identified host factor that assists in HCV secretion. These results demonstrate the critical function of GP73 in HCV secretion and provide new insights into the therapeutic design of antiviral strategies. Hepatitis C virus (HCV), which is a major cause of chronic liver diseases, affects approximately 170 million people worldwide. More than 80% of infected patients develop chronic infection and one-third of these patients develop progressive liver injury, fibrosis, cirrhosis and hepatocellular carcinoma (HCC) over a period of 20 to 30 years Hepacivirus genus of the Flaviviridae family, which also includes several classic flaviviruses, such as dengue virus and yellow fever virus HCV, a small enveloped RNA virus, belongs to the cis- and medial- Golgi cisternae Recent studies have revealed the importance of Golgi components in HCV maturation. Protein kinase D (PKD) is recruited to the trans-Golgi network to influence vesicular trafficking to the plasma membrane. The expression of HCV proteins inactivates PKD to enhance HCV secretion To determine the correlation between GP73 and HCV infection at the cellular level, the effect of GP73 on the HCV life cycle and its regulation in HCV-infected cells were examined in this study. Our results suggest that GP73 was upregulated by HCV infection, and GP73 could enhance HCV virion secretion by increasing apolipoprotein E (APOE) and interacting with APOE. Our results confirm the connection between GP73 and HCV infection at the molecular and cellular levels for the first time, and provide new insights into the therapeutic design of anti-HCV strategies.GP73 truncation expression plasmids were generated as previously described GP73 into the lentiviral vector pRlenti. Plasmids pSuper-shGP73-1 and pSuper-shGP73-2, which were used to generate siRNA against GP73, were constructed by inserting the target sequence into the pSuper  in site with Bgl II and Hind III. The inserted sequences are as follows:shGP73-1:5'-GATCCCCGTTGAGAAAGAGGAAACCAATTTCAAGAGAATTGGTTTCCTCTTTCTCAACTTTTTA-3'.shGP73-2:5'-GATCCCCCGAATAGAAGAGGTCACCAAATTCAAGAGATTTGGTGACCTCTTCTATTCGTTTTTA-3'.The target sequences against GP73 are underlined. The glycoprotein sequences of HCV J6 and JFH1 were cloned into pCDNA3.1 to generate pJ6E1E2 and pJFH1E1E2 for HCVpp production as previously reported Huh7, Huh7.5.1, and HEK 293T cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum , 100 units/mL penicillin, and 0.1% (w/v) streptomycin. pSGR-Con1 and pSGR-JFH1 RNA were transfected into Huh7 and Huh7.5.1 cells, respectively. HCV SGR-harboring cells (Huh7-SGR and Huh7.5.1-SGR) were subsequently established as previously described HCV core monoclonal antibody (C7-50) and NS3 monoclonal antibody (H23) were purchased from Abcam (USA). Anti-FLAG monoclonal antibody M2 was purchased from Sigma (USA). Rabbit polyclonal antibody anti-APOE was purchased from Sino Biological Inc. . Anti-GP73 monoclonal antibody (5B12) and rabbit polyclonal antibody were prepared in our laboratory.in vitro RNA transcription, transfection into Huh7.5.1, and preparation of HCV-GFP viruses were all performed as previously reported The HCVpp was prepared as previously reported To produce infectious GP73 and shGP73 lentiviral particles, HEK293T cells were co-transfected with either lentiviral vector pRLenti-GP73 or pSuper-shGP73-1/2 together with packaging plasmid psPAX2 (Addgene plasmid 12260) and pMD2.G (Addgene plasmid 12259). At 48 h after transduction, the virus-containing supernatant was filtered through 0.45 µm filters, and incubated with the target cells in the presence of 4 µg/mL polybrene .Cells were seeded onto cover slides 24 h after transfection or 48 h after infection. After overnight culture, slides were directly fixed in 4% paraformaldehyde in PBS for 15 min, followed by permeabilization with 0.5% Triton X-100 for 5 min. These cells were then stained in diluted antibodies. The FLAG epitope in the fusion proteins was detected using anti-FLAG monoclonal antibody M2 , followed by goat anti-mouse immunoglobulin conjugated to Alexa Fluor 488 dye . APOE was detected using anti-APOE rabbit polyclonal antibody and a goat anti-rabbit immunoglobulin conjugated to Alexa Fluor 488 dye . Endogenous GP73 was detected using anti-GP73 monoclonal antibody (5B12) or rabbit polyclonal antibody prepared in our laboratory. The slides were viewed and imaged using a confocal microscope .The culture medium of HCV-infected cells was centrifuged to remove cellular debris and filtered through 0.45 µm filters. The supernatant was pelleted by centrifugation at 100,000 ×g for 3 h at 4°C. The pellet was resuspended in 400 µL PBS buffer and applied on a 20%–60% sucrose gradient (3.5 mL volume) in SW60 tubes (Beckman Coulter) and centrifuged at 100,000 × g (RCFav) for 16 h at 4°C. We collected 340 µL fractions from the top of the gradient. The fractions were tested for protein levels using western blot and relative viral titer with a flow cytometer.For intracellular GP73 and APOE interaction, 293T cells were cotransfected with plasmids expressing FLAG-tagged GP73 and HA-tagged APOE. At 30 h after transfection, cells were harvested and lysed in freshly prepared immunoprecipitation buffer consisting of 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 0.5% NP40 and protease inhibitor cocktail (Sigma) for 20 min on ice, followed by centrifugation at 16,000 ×g for 30 min at 4 °C. The soluble supernatants were processed for immunoprecipitation by incubation with specific antibodies or normal mouse IgG control overnight at 4 °C. Protein A/G beads (Amersham Bioscience) were added for an additional 3 h. The immunoprecipitants were washed eight times with PBS. Associated proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies.For secreted GP73 and APOE interaction, the culture medium was collected 48 h after transfection and centrifuged to remove cellular debris. The supernatants were concentrated by an Amicon Ultra -15 centrifugal filter  and processed for immunoprecipitation as described above.The total RNA was extracted from the cells using a Total RNA Isolation Kit  or from the supernatants using QIAamp Viral RNA Mini Kit  according to the manufacturer’s instructions. First-strand cDNA was synthesized using RevertAid reverse transcriptase  and qRT-PCR was performed using Premix Ex Taq  in a CFX96 Real-Time PCR Detection System . The primers used in this study are listed in 4 naive Huh7.5.1 in a 24-well plate for 6 h. At 72 h post-infection, the infected cells were detected using a flow cytometer . The intracellular and supernatant HCV titers were represented as relative titers calculated as percentages of the GFP-positive rate of the control, which corresponded to the absolute HCV titer . Statistical analysis was performed using GraphPad Prism software . P<0.05 was considered to be statistically significant. did not increase intracellular HCV titer or viral RNA , which sMany new host cofactors required in HCV egress were recently identifiedTo determine whether GP73 and APOE interact directly, co-IP assays were performed. As indicated by The overall results suggest that one underlying mechanism of GP73 enhanced HCV production is through the upregulation of APOE and its assistance in HCV secretion through interaction with APOE.Serum GP73 is considered as a biomarker for liver diseases with clinically proven sensitivity and specificity To investigate whether HCV infection upregulates GP73 expression at the cellular level, we detected the change in expression of GP73 in HCV SGR-harboring cells and HCV-infected cells. The mRNA and protein expression levels of GP73 significantly increased in SGR-harboring cells and declined when the HCV genome was cleared, either in Huh7 or Huh7.5.1 cells . The expThe upregulation of GP73 in SGR-harboring cells indicates that GP73 was probably upregulated by viral RNA replication and non-structural protein expression. However, no evident change in the expression of GP73 was observed in stably cells expressing non-structural proteins . This fiRecent studies suggest the involvement of the Golgi apparatus in HCV secretion APOE, a previously defined HCV secretion related gene, was upregulated by GP73. The increase in the intracellular and supernatant APOE in GP73-overexpressed cells indicates that the upregulation of GP73 enhanced the expression and secretion of APOE . Recent APOE allele epsilon 4 is closely related to Alzheimer’s disease In summary, we demonstrate that GP73, a novel biomarker for liver diseases, is upregulated by HCV infection and enhances HCV secretion. In addition, we illustrate the possible mechanism by which GP73 increases HCV secretion. APOE, an identified host factor that facilitates HCV secretion, is upregulated by GP73, and can interact with GP73. The study is the first to confirm the correlation between GP73 and HCV infection at the cellular level. The identification of the involvement of GP73 in the process of HCV secretion strengthens the key role of the Golgi in HCV secretion and provides new insights into the therapeutic design of anti-HCV strategies and physiological function of GP73.Figure S1Infection efficiency measured by flow cytometry is highly coincident to the HCV titer. Naive Huh7.5.1 cells were incubated with HCV-containing supernatant at different volumes for 6 h before washout. Then, infectivity was detected by flow cytometry at 72 h post-infection.(TIF)Click here for additional data file.Figure S2A) GP73 mRNA level in stable cells. (B) Infection efficiency of HCV-GFP in stable Huh7 cells at 0.02 MOI. (C) Intracellular viral RNA level. (D) Supernatant infectivity at 96 h post-infection. The results were presented as mean ± the SEM.GP73 enhances HCV production in stable Huh7 cells. Stable GP73 overexpressed cells (Huh7 GP73) and GP73 knockdown cells (Huh7 shGP73) were established with lentivirus as described in the “Materials and Methods” section. ((TIF)Click here for additional data file.Figure S3B) Huh7 cells that stably express HCV non-structural protein were established with lentivirus infection and puromycin screening. GP73 mRNA levels were quantified by qRT-PCR. (N.S.: not significant).GP73 is not upregulated by HCV non-structural protein expression. (A) Huh7 cells were transfected with indicated plasmids. GP73 mRNA level was quantified by qRT-PCR at 72 h post-transfection. ((TIF)Click here for additional data file.Table S1Sequences of qRT-PCR primers used in this study.(DOCX)Click here for additional data file.


Nucleosome translocation along DNA is catalyzed by eukaryotic SNF2-type ATPases. One class of SNF2-ATPases is distinguished by the presence of a C-terminal bromodomain and is conserved from yeast to man and plants. This class of SNF2 enzymes forms rather large protein complexes that are collectively called SWI/SNF complexes. They are involved in transcription and DNA repair. Two broad types of SWI/SNF complexes have been reported in the literature; PBAF and BAF. These are distinguished by the inclusion or not of polybromo and several ARID subunits. Here we investigated human SS18, a protein that is conserved in plants and animals. SS18 is a putative SWI/SNF subunit which has been implicated in the etiology of synovial sarcomas by virtue of being a target for oncogenic chromosomal translocations that underlie synovial sarcomas.We pursued a proteomic approach whereby the SS18 open reading frame was fused to a tandem affinity purification tag and expressed in amenable human cells. The fusion permitted efficient and exclusive purification of so-called BAF-type SWI/SNF complexes which bear ARID1A/BAF250a or ARID1B/BAF250b subunits. This demonstrates that SS18 is a BAF subtype-specific SWI/SNF complex subunit. The same result was obtained when using the SS18-SSX1 oncogenic translocation product. Furthermore, SS18L1, DPF1, DPF2, DPF3, BRD9, BCL7A, BCL7B and BCL7C were identified. ‘Complex walking’ showed that they all co-purify with each other, defining human BAF-type complexes. By contrast,we demonstrate that human PHF10 is part of the PBAF complex, which harbors both ARID2/BAF200 and polybromo/BAF180 subunits, but not SS18 and nor the above BAF-specific subunits.SWI/SNF complexes are found in most eukaryotes and in the course of evolution new SWI/SNF subunits appeared. SS18 is found in plants as well as animals. Our results suggest that in both protostome and deuterostome animals, a class of BAF-type SWI/SNF complexes will be found that harbor SS18 or its paralogs, along with ARID1, DPF and BCL7 paralogs. Those BAF complexes are proteomically distinct from the eukaryote-wide PBAF-type SWI/SNF complexes. Finally, our results suggests that the human bromodomain factors BRD7 and BRD9 associate with PBAF and BAF, respectively. Drosophila by brahma and in humans by BRM and BRG1.Gene expression programs determine cell identity and response to endocrine stimuli, as has been demonstrated most dramatically by the generation of induced pluripotent stem cells with the Oct4, Sox2, Klf4 and c-Myc transcription factors The C-terminal bromo domain-bearing SNF2 enzymes are found in so-called SWI/SNF multiprotein complexes and are conserved in most eukaryotes. They are implicated in transcriptional regulation and multiple DNA repair pathways In mice and humans, at least 20 different SWI/SNF complex subunits have been reported  [10], [3flox/flox mouse is the most lethal tumor suppressor mouse model reported to date Strikingly, multiple SWI/SNF subunits function as tumor suppressors in man and mouse, adding a key medical dimension to SWI/SNF research Another link to cancer is provided by the SS18-SSX oncofusion proteins bona fide BAF-type SWI/SNF complex subunits.We found SS18 to be present in BAF-class human SWI/SNF chromatin remodeling complexes. Purification of SS18-SSX1 revealed that this oncofusion protein resides in the same complexes. Interestingly, we detected several additional putative SWI/SNF interactors In order to define the protein complexes harboring known and suspected human SWI/SNF subunits we generated stable human embryonic kidney cell (Hek293) clones transduced with retroviral TAP-tag fusion expression constructs TAP has consistently been comparatively low. For instance, in most INI1TAP preparations we detect BRG1 but not BRM, and ARID1A but not ARID1B  results in production of the oncogenic SS18-SSX1 protein fusion it was of interest to compare the proteomic environments of SS18 and the SS18 oncofusions. Essentially, purification of SS18-SSX1 protein . All sub protein . We concDPF2, also known as ubi-d4 or Requiem, is ubiquitously expressed and implicated in apoptosis TAP purification results indicate that DPF2 resides mainly in ARID1-bearing BAF complexes, since no polybromo or ARID2 peptides were identified, whilst high confidence ARID1A and ARID1B peptides were detected  again pointing towards the BAF variants of SWI/SNF. Finally, like DPF2TAP, BCL7CTAP also pulled down SS18l1 . However, BRD9TAP did yield significant mass spectrometry results  or only in animals  .Drosophila melanogaster except that there is only one ARID1 ortholog, namely OSA Essentially there are two types of human SWI/SNF complexes Drosophila SAYP Our mass spectrometry analysis of affinity tag-mediated protein complex purifications confirms bipartition of SWI/SNF complexes in BAF and PBAF-class complexes. We demonstrate here that the paralogous cancer-related minor SWI/SNF subunits DPF1, -2, -3; BCL7A, -B, -C; and SS18 and SS18L1 reside in BAF-class human SWI/SNF complexes and, that PHF10 marks PBAF SWI/SNF complexes. Moreover, because quantitative analysis indicates that the chief interaction partners of PHF10, DPF2, SS18, SS18-SSX1, BCL7A and BCL7C are the other SWI/SNF subunits, we speculate that they exert their molecular action through their respective SWI/SNF complexes. It remains to be seen indeed to what extent our results, which were obtained in one human cell line, can be extrapolated to other cell types and even other organisms. Considering the congruence between our data and a recent studies on Since we could not detect notable differences between SS18 and its oncogenic fusion products at the proteomic level, the oncogenic activity of the SS18-SSX fusions may have to be sought either at the level of SWI/SNF (dis)assembly dynamics, post-translational modifications or an affinity for specific genomic loci TAP, SS18TAP and the SS18-SSX1TAP were used as affinity bait and since in the reciprocal experiment, BRD9TAP purifications contained SS18 and BCL7A. We note however that BRD9TAP did not efficiently pull down BAF proteins in our experimental set-up  and SS18-SSX1  coding sequence were amplified by PCR from pIRES2 vectors cgtatcGAATTCATGGCGGCTGTGGTGGAGAAT, tgtcttCTCGAGTCAAGAGGAGTTCTGGTTCTGGTA), BCL7C , BRD9  and PHF10  coding sequences were amplified from cDNA clones (RZPD or OriGene). BCL7A  was amplified from cDNA of Hek293 cells. INI1  was amplified from pUHD-10-3-INI1 MYC epitope (GAGCAGAAGCT TATCAGCGAGGAGGACCTGGGCGGGGAATTCGCCGGCAAGCCCCGGCATATGAATTTAATG) or, in the case of PHF10, a TY1 epitope (GAGGTGCACACCAACCAGGACCCCCTGGACGAATTCGCCGGCGCCGATGCCGGCAAGCCCCGGCA TAGGACCGGT) 5′ of the EcoRI cloning site. Every clone was verified by DNA sequencing.Tandem Affinity Purification (TAP) constructs were generated by PCR using the oligomers indicated in parentheses and cloned into the XhoI and EcoRI restriction sites in the retroviral expression vector pZXN, whereby the TAP-tag sequence was fused to the coding sequences at their N-terminus 2. Retroviral stable cell lines were generated as previously described Human Embryonic Kidney  and phoenix cells were grown in Dulbecco's modified Eagles medium (Invitrogen) supplemented with 10% FCS, penicillin 100 µg/ml and streptomycin 100 U/ml (Invitrogen) at 37°C in 5% COTandem Affinity Purification was performed as previously described in detail Co-immunoprecipitations were performed on TEV cleavage eluates obtained as described above, using antibodies directed against BRM (Abcam 15597), DPF2 (Aviva systems biology ARP33221_P050) or BRD9 (Aviva systems biology ARP34803_T200), under the same conditions as the anti-MYC or TY1 immunoprecipitations in the TAPtag purification protocol. The immunoprecipitated proteins were separated by SDS-PAGE, transferred onto nylon filters and probed with anti-MYC antibodies, which recognize the transduced SS18-SSX1  or SS18 http://www.msquant.sourceforge.net) was used to parse the raw files and for generation of peak lists. The mascot algorithm was used to identify the proteins The silver stained gel lanes were cut into small pieces. After reduction and alkylation the proteins were trypsin (Promega) digested and extracted from the gel using trifluoroacetic acid (TFA). Peptides were sequenced using a nano-high-pressure liquid chromatography Agilent 1100 nanoflow system connected online to a 7-Tesla linear quadrupole ion-trap Fourier transform (FT) mass spectrometer  essentially as described previously Expression profiling was performed on four Hek293 polyA mRNA samples by microarray analysis using Affymetrix human exon array 1.0 ST according to manufacturer instructions .Data S1Mass spectrometry results, including; accession numbers, short protein descriptions, peptide sequences, associated Mascot score, peptide delta score and absolute calibrated mass relative error.(XLS)Click here for additional data file.Data S2Quadruplate polyA mRNA expression profile of Hek293 cells determined with the Affymetrix human exon array 1.0 ST platform.(XLS)Click here for additional data file.