In this study, we generated a series of C-terminal mutants of D1R that were tagged with or without enhanced yellow fluorescent protein, and analyzed the consequences of these mutants on the plasma membrane trafficking of D1R and cyclic AMP response to D1R stimulation. D1R with mutations within the endocytic recycling signal (amino acid residues 360–382) continued to be functional, albeit decreased relative to wild-type D1R. Mutation of the palmitoylation site (347C>S) of D1R did not impair its trafficking to the plasma membrane, but abolished its ability to increase cyclic AMP accumulation. In contrast, replacement of di-leucines (344–345L>A) by alanines resulted in the retention of D1R in the early endosome, decreased its glycosylation, and prevented its targeting to the plasma membrane. Our studies suggest that di-L motif at the C-terminus of D1R is critical for the glycosylation and cell surface targeting of D1R.The dopamine D Their trafficking to the plasma membrane is needed for response to their extracellular ligand. Therefore, the appropriate delivery of GPCRs to the plasma membrane permits receptor/ligand interaction. Their subsequent internalization and re-insertion to the plasma membrane are of fundamental importance in the regulation of GPCR activity.1R plays an important role in its plasma membrane trafficking. Vargas and von Zastrow 1R. Bermak et al. 333XXXF337XXXF341, which is highly conserved among GPCRs, functioned independently as an endoplasmic reticulum (ER)-export signal for the D1R. It was further demonstrated that F337(X)6L344L345 plays a role in ER export of several GPCRs, including α1B-AR, α2B-AR, AT1R, and β2-AR 1AR gets stuck in ER, indicating that the C-terminal di-leucine motif is involved in the proper folding of 5-HT1AR Several studies have shown that the C-terminus of D1R from the ER to the plasma membrane, we generated a series of C-terminal mutants of D1R and analyzed their trafficking and function following agonist stimulation. Our results indicated that di-L motif is critical for the plasma membrane targeting of D1R. However, the internalized D1R continues to be functional, if stimulated by a cell permeable agonist.To characterize further the structural determinants involved in the trafficking of D1R was amplified by PCR with Hind III digestion site at the N-terminus and Sac II digestion site at the C-terminus, and then sub-cloned into the mammalian expression vector pEYFP-N1  to generate pYG1 (pEYFP-hD1R). Then pYG1 was utilized in producing C-terminal mutants of D1R (pYG2-pYG16)  , using tAll constructs generated and used in this study  were con2. For transient transfections, cells were grown on the coverslips in 6-well plates and transfected at a ratio of 1 µg of plasmid DNA to 5 µL Lipofectamine 2000  in 200 µL of serum-free medium, according to the manufacturer's protocol. The cells were processed ∼36 h after transfection for confocal microscopy or western blotting. Transfection efficiency was estimated to be same for each transfected plasmid based on the YFP fluorescence, using an epifluorescence microscope.Human embryonic kidney (HEK) 293 cells  were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum  at 37°C with 95% air and 5% COAfter transfection for ∼36 hours, HEK 293 cells grown on coverslips were rinsed in cold PBS and fixed with 4% paraformaldehyde for 20 min on ice, and then permeabilized with methanol for 20 min on ice. The following primary antibodies were used: monoclonal mouse anti-GM130 and monoclonal mouse anti-calnexin , and polyclonal rabbit anti-EEA1 . Alexa Fluor 568 donkey anti-mouse IgG and Alexa Fluor 568 donkey anti-rabbit IgG were purchased from Invitrogen. Mounting medium with DAPI  was used. Images were obtained on Olympus Fluoview FV300 laser scanning confocal microscope equipped with a 40X 1.4 N.A. objective. FITC filter was used for imaging YFP-tagged proteins and the green color was selected as the pseudo color for YFP-tagged proteins in the figures. Images were processed using Adobe Photoshop. At least four studies were performed for any particular transfection. About 300 randomly selected transfected cells per coverslip were observed. The colocalization analysis was performed using Image J software.1R or YFP-di-L D1R plasmid for cell surface biotinylation and isolation studies, using a kit from Thermo Scientific Pierce . In this study, one 10-cm dish of cells for each plasmid was used. Twenty-four hours after transfection the media were aspirated and cells quickly washed twice with 5 mL of ice-cold PBS per dish. Then, 5 mL of the biotin solution were added to each dish and gently agitated for 30 min at 4°C. At the end of 30 min, the reaction was stopped by the addition of 500 µL of quenching solution. The rest of the steps followed the manufacturer's protocol. The samples were subjected to SDS-PAGE, and then immunoblotted with anti-GFP antibody.HEK 293 cells were cultured in 10-cm dishes and transfected with YFP-wild-type (wt) DCells grown in 6-well plates were washed twice with ice-cold PBS and then lysed in cold RIPA buffer containing 1 mM DTT and protease and phosphatase inhibitors  on ice for 10 min. The cells in each well were scraped and transferred to a 2 ml centrifuge tube. The lysates were centrifuged at 12,000 rpm in a pre-cooled centrifuge for 15 min and the supernatants were transferred to new tubes. The lysates were subjected to SDS-PAGE (4–12% gradient gel from Invitrogen) and immunoblotted with specific antibodies. Li-Cor Odyssey infrared imaging system  was used for western blotting detection.1R antibody was from Origene . Secondary antibodies were all purchased from Li-Cor Biosciences .Goat polyclonal anti-GFP antibody purchased from Abcam , which is designed to recognize all variants of GFP, was used for blotting all YFP-tagged proteins in this study. Rabbit anti-D1R), pYG2 (YFP-di-L D1R), pYG15 (wt D1R), and pYG16 (di-L D1R). After an 8 hr transfection, the cells were treated with or without tunicamycin  for another 16 hr. Then the cells were collected for western blotting following the steps mentioned above. To determine the oligosaccharides in the glycosylated D1R, 36 hr post-transfection, the cells were collected and denatured in glycoprotein denaturing buffers  at 100°C for 10 min. Denatured sampled were digested with 500 U endoglycosidase H or peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F (New England Biolabs) by incubation at 37°C for 1 hr. After digestion, samples were subjected to western blotting as mentioned above to analyze their state of glycosylation.HEK 293 cells grown in the 6-well plates were transfected with same amount of pYG1  fenoldopam  , or the cell-membrane impermeable D1R agonist (in the absence of D5R), A-68930 hydrochloride  . The cells were concomitantly treated with a phosphodiesterase inhibitor, IBMX . After the 15 min incubation period, 300 µL of 0.1 M HCl were added and incubated for another 20 min at room temperature. The amount of cAMP in each well containing ≥ 1 mg protein/ml was measured by an immunoassay kit , according to the manufacturer's protocol. Each treatment was performed in triplicate.HEK 293 cells were cultured in 6-well plates and then transfected with different Dt test. A P value < 0.05 was considered significant.Data were expressed as mean + standard error. Significant differences between two groups were determined by Student's 1R contains a di-leucine motif (L344/345) that is very close to the 7th transmembrane domain (TMD 7), two palmitoylation sites (C347/351), and an endocytic recycling signal sequence (T360—S382) identified by Vargas and von Zastrow 1R. To study further the role of the C-terminus of D1R in its cellular trafficking and function, the di-leucine motif (L344/L345) and a series of amino acids within the endocytic recycling signal were mutated to alanine residues using site-directed mutagenesis; one palmitoylation site cysteine (C347) was replaced by a serine residue using the same technique , which could be the aggregation of the proteins. When blotted with anti-GFP antibody (1R and di-L mutant had a ∼30 kDa that may represent the soluble YFP (27kDa). The bands of ∼40–50 kDa were probably degraded D1Rs. We have previously reported 1R to the plasma membrane 1A which is poorly glycosylated does not traffic to the plasma membrane 1R may not be fully glycosylated and fails to be targeted to the cell surface, suggesting that di-leucine motif in the C-terminus of D1R plays an important role in its plasma membrane trafficking.We have studied the function of dopamine receptors in the kidney antibody , both YF1R could traffic to the plasma membrane, we studied the cell trafficking of cell surface-biotinylated pYG1 (pEYFP-wt D1R) or pYG2 (pEYFP-di-L D1R). Briefly, HEK 293 cells were transfected with YFP-wild-type (wt) D1R or YFP-di-L D1R plasmid for cell surface biotinylation and isolation studies using a kit from Thermo Scientific Pierce. Twenty-four hours after transfection, the media were aspirated and cells quickly washed twice with 5 mL of ice-cold PBS per 10-cm dish. Then, 5 mL of the biotin solution were added to each dish and gently agitated for 30 min at 4°C. The reaction was stopped by the addition of 500 µL of quenching solution. The rest of the steps followed the manufacturer's protocol. The samples were subjected to SDS-PAGE, and then immunoblotted with anti-GFP antibody.To investigate further whether or not di-L mutant D1R  than YFP-di-L D1R proteins  were eluted down after cell surface biotinylation and isolation. In other words, YFP-wt D1R protein was mostly expressed at the cell surface, but not the YFP-di-L D1R protein. This result is consistent with the fluorescence microscopy studies shown in As shown in 1R plays a critical role in the plasma membrane targeting of D1R. The cell surface isolation results also suggested that the protein trafficked to the plasma membrane is glycosylated, consistent with our previous reports Therefore, these results indicated that di-L motif in the C-terminal of D1R was mainly localized in the intracellular compartments, we determined the identity of these compartments. We immunostained pYG2 (di-L mutant D1R)-transfected HEK 293 cells with the cis-Golgi marker, GM130, the endosomal marker, EEA1, and the ER marker, calnexin. Di-L mutant D1R did not colocalize with GM130 . YFP-wt D1R migrated as a band of ∼90 kDa and a band of ∼70 kDa in untreated cells -treated YFP-di-L D1R cells  to remove both high-mannose and complex N-glycosylation (1R) was still visible in both wt and di-L mutant D1R, suggesting that the D1R was not totally high-mannose N-glycosylated. By contrast, PNGase F treatment resulted in the migration of both wt D1R and di-L mutant D1R at the ∼60 kDa, which corresponded to unglycosylated non-tagged D1R protein, indicating that both wt and partial di-L mutant D1R goes through complex glycosylation, which occurs only in the Golgi apparatus It has been known that the majority of mature glycoproteins traversing the Golgi are N-glycosylated, whereas those residing in the ER are high-mannose glycosylated sylation . After E1R and other C-terminal mutants, we measured the accumulation of cyclic AMP (cAMP) in the transfected cells that were treated with vehicle (water) or fenoldopam, D1R agonist. To achieve similar transfection efficiency, HEK 293 cells were transfected with same amount of each plasmid listed in To test the functionality of di-L mutant D1R or D5R (data not shown). Therefore, as we expected, fenoldopam  did not affect the cAMP accumulation in untransfected HEK 293 cells (1R (pYG1)-transfected HEK 293 cells had a marked increase in cAMP accumulation with fenoldopam stimulation (1R (pYG2)-transfected cells in response to fenoldopam was similar to wt D1R, although this mutant failed to traffic to the cell surface  to increase cAMP accumulation in these cells  in which the mutated sites were within the endocytic recycling signal had an increase in cAMP accumulation in response to fenoldopam stimulation, albeit to a much lower extent than those observed in wt D1R. These results could be taken to suggest that these mutants could be internalized as the wt after the agonist stimulation, but are minimally recycled back to the plasma membrane and therefore, respond minimally to continuous agonist stimulation, thus the increase in cAMP was much lower than that of the wt. However, this is unlikely because di-L mutant D1R which is minimally trafficked to the plasma membrane had a marked increase in cAMP response to the cell membrane permeable but not cell membrane impermeable agonist. Rather, these mutations interfered with the intrinsic ability of D1R to respond to agonist stimulation, the mechanisms of which remain to be determined.When palmitoylation site (Cys347) was mutated to serine residue (347 C>S), accumulation of cAMP was not increased by fenoldopam, indicating that palmitoylation of Dt action , pYG8. T1R traffics in the cell and how its trafficking relates to the function, although it has been reported that the C-terminus of D1R is very important for its trafficking and function In general 1Rs in transfected HEK 293 cells. Our results interestingly showed that when the di-leucine motif (L344-345) at the C-terminus of D1R was mutated, the mutant protein was not able to traffic to the plasma membrane; instead, it was localized in the early endosome. This data suggested that this C-terminal di-L motif is a plasma membrane targeting signal. Kim et al. 1R (truncated at position 347) was diminished relative to the wild-type. In their mutant, di-L motif was intact, but the mutant could not get to the cell surface. Combined with our results, we propose that the di-L motif (L344-345) is required for the cell surface targeting of D1R, but is not the only signal for this trafficking. This C-terminal di-L motif is highly conserved in GPCRs, but replacement of di-leucine motif in the C-terminus of the β2-AR by alanines resulted in a marked reduction in internalization 2-AR, which is similar to its function in other membrane proteins In this study, we investigated the plasma membrane trafficking and function of a series of C-terminal mutant D1R has two palmitoylation sites at positions 347 and 351 in the carboxyl tail 1R in maintaining the conformation for antagonist binding and is essential for D1R's agonist-induced desensitization, however, the pharmacological and functional properties of C351S mutant were similar to that of wild-type D1R 1R trafficking and responsiveness Another common structural theme among GPCRs is palmitoylation of one or more sites of the C-terminal tail near the seventh transmembrane domain 1R deceased the cell surface trafficking of D1R et al.1R localization to the plasma membrane. The reason(s) for this discrepancy is not clear. In this study, we found that the glycosylation of YFP-di-L D1R was markedly decreased and markedly limited expression at the cell surface compared with the wild-type  without regard to type, whereas Endo H removes only high mannose and some hybrid types of N-linked carbohydrates. The results in 1R is that of complex N-glycosylation. This glycosylation state may explain why YFP-di-L D1Rs  is relatively cell-membrane permeable we hypothesized that fenoldopam could bind to the di-L D1Rs that continue to be functional inside the cell. However, when the cells were treated with A-68930 , which is a relatively cell-membrane impermeable, the accumulation of cAMP was not affected. These data indicated that the response of di-L mutant D1R to extracellular agonist stimulation was impaired because of a failure of di-L mutant D1R to be trafficked to the plasma membrane. The mutants other than pYG2 had a limited cAMP response to both fenoldopam and A-68930, probably because of impairment of function, not necessarily related to cell surface membrane trafficking. It could be that they have greatly diminished resensitization or they cannot find Gs and/or cylase in early/recycling endosomes.The function of each mutant D pYG2 in , which w1R is required for its plasma membrane trafficking and glycosylation. Further investigations may reveal how di-L motif is involved in the sorting of D1R.In summary, di-L motif (L344-345) at the C-terminus of DFigure S1Western blots using anti- D1R antibody. HEK 293 cells were transfected with YFP-wt D1R and YFP-di-L D1R plasmids in 6-well plates. 36 hours after transfection, cells were washed twice with ice-cold PBS and then lysed in cold RIPA buffer containing 1 mM DTT and protease and phosphatase inhibitors on ice for 10 min. The cell lysates were subjected to SDS-polyacryalmide gel electrophoresis (4–12% gradient gel) and immunoblotted with rabbit anti-D1R antibody.(TIF)Click here for additional data file.


Viruses have to encapsidate their own genomes during the assembly process. For most RNA viruses, there are sequences within the viral RNA and virion proteins needed for high efficiency of genome encapsidation. However, the roles of host proteins in this process are not understood. Here we find that the cellular DEAD-box RNA helicase DDX6 is required for efficient genome packaging of foamy virus, a spumaretrovirus. After infection, a significant amount of DDX6, normally concentrated in P bodies and stress granules, re-localizes to the pericentriolar site where viral RNAs and Gag capsid proteins are concentrated and capsids are assembled. Knockdown of DDX6 by siRNA leads to a decreased level of viral nucleic acids in extracellular particles, although viral protein expression, capsid assembly and release, and accumulation of viral RNA and Gag protein at the assembly site are little affected. DDX6 does not interact stably with Gag proteins nor is it incorporated into particles. However, we find that the ATPase/helicase motif of DDX6 is essential for viral replication. This suggests that the ATP hydrolysis and/or the RNA unwinding activities of DDX6 function in moderating the viral RNA conformation and/or viral RNA-Gag ribonucleoprotein complex in a transient manner to facilitate incorporation of the viral RNA into particles. These results reveal a unique role for a highly conserved cellular protein of RNA metabolism in specifically re-locating to the site of viral assembly for its function as a catalyst in retroviral RNA packaging. Foamy viruses are complex retroviruses that infect non-human primates, cats, cows, and horses. Humans are not natural hosts but can acquire primate foamy viruses as zoonotic infections. During foamy virus assembly process, viral RNAs and Gag capsid proteins are targeted to a discrete intra-cytoplasmic site where viral particles are assembled. One key step in this process is to effectively incorporate the virus genome into particles. For retroviruses, encapsidation of viral genomic RNA is known to initiate when specific packaging sequences within the viral RNA are recognized by the nucleocapsid domain of the Gag polypeptide. However, the contribution of host factors to the assembly process is largely unknown. In this study, we find that after foamy virus infection some of the cellular DEAD-box RNA helicase DDX6 specifically re-localizes to the viral assembly site, and is needed for efficient packaging of viral RNA into particles. Our data suggest that the ATP hydrolysis and RNA unwinding activities of DDX6 function in remodeling the structure of viral RNA and/or RNA-Gag ribonucleoprotein to facilitate its incorporation into particles. Our work provides the first report of an evolutionarily conserved host protein involved in the assembly of retrovirus genomes into particles. Spumaretrovirinae, are complex retroviruses that infect all non-human primates, cats, cows, and horses. Humans are not natural hosts but can acquire primate foamy viruses as zoonotic infections cis-acting packaging signal in the viral RNA is recognized by the nucleocapsid domain of Gag. While packaging sequences in the RNA and protein motifs in the nucleocapsid protein are well studied, the contribution of host factors to the process of viral RNA packaging is largely unknown.Foamy viruses, the only genus in the retrovirus subfamily In eukaryotic cells, translationally repressed mRNAs are often sequestered in discrete cytoplasmic RNA granules called P bodies, where they are either degraded or stored for later translation. If mRNAs are stalled in a translation preinitiation complex , they are packaged into another form of granules named stress granules. Components of P bodies and stress granules are closely associated with RNA metabolism. P bodies include components of the 5′-3′ mRNA degradation pathway such as Dcp1, Dcp2, Ago1, Ago2, MOV10, and DDX6  occurs mainly in the cytoplasm near the MTOC where Gag capsid protein accumulates nd siRNA transfection, cells were infected with PFV for 48 hrs, and culture supernatants were analyzed by the FAB assay to measure infectious titers Six P body and stress granule proteins were individually silenced using gene-specific siRNA to examine whether or not they were required for replication of prototype foamy virus (PFV). PFV was originally isolated from a human tumor cell line, but is now known to be a chimpanzee foamy virus that was zoonotically transmitted to the human from whom it was cultured For yeast retrotransposons Ty1 and Ty3, their viral RNAs, Gag proteins, and virus-like particles all accumulate in P bodies where particle assembly and RNA packaging occur in vivo, we adapted a pumilio-based bimolecular fluorescence complementation (BiFC) reporter system developed by Tilsner et al.gag, resulting in minimal changes from the wild-type sequences. Consequently, all viral genomic RNAs as well as unspliced gag and singly spliced pol mRNAs contained UGUAAAUA and UGUAGAUA in their sequences    where Gantibody) . Interesntibody) . These rTo identify the role of DDX6 in viral replication, we examined viral RNA and proteins in both intracellular and extracellular fractions from DDX6-knockdown cells. When cells were transfected twice with 120 nM siRNA as shown in To understand the specific role of DDX6 in the assembly of virus genome, we wanted to find out whether DDX6 interacted with the viral RNA or Gag capsid proteins. Although DDX6 co-localized with Gag at the assembly site, it was unclear if DDX6 bound to Gag proteins directly. Cell lysates from pcPFV-transfected 293T cells were immunoprecipitated using anti-DDX6 or anti-Gag antibody. Because Dcp1 was known to physically interact with DDX6 In vitro, DDX6 has RNA-dependent ATPase and ATP-dependent RNA helicase activities that are determined by the highly conserved DEAD box as well as sequences in its C-terminal domain In this study, we find that the RNA helicase DDX6, a cellular protein associated with P bodies as well as stress granules, is re-directed to the site of foamy virus assembly and plays an important role in assembly of viral RNA into particles. An intact ATPase/helicase domain of DDX6 is essential for viral replication, suggesting that the ATP hydrolysis and RNA unwinding activities of DDX6 function in rearranging the structure of viral RNA and/or RNA-Gag complex into a proper conformation to facilitate viral RNA packaging.cis-acting packaging sequences in the viral RNA to promote its recognition by Gag. More studies will be needed to clarify these interactions and to understand the exact role of DDX6 in the process of viral RNA encapsidation.About 20% of endogenous DDX6 proteins, but not other P body/stress granule proteins tested, are re-located to the viral assembly site near the MTOC where DDX6 co-localizes with viral RNA and Gag capsid proteins. We have ruled out a possible role of DDX6 in transporting viral RNA and Gag to the MTOC area, and it is unclear how DDX6 is re-localized to the assembly site which occurs specifically only after virus infection or transfection. DDX6 could bind to Gag protein and/or viral RNA and be transited to the MTOC area. However, we have no evidence for a direct, stable interaction between DDX6 and Gag . The obsin vivo are complex. Although generally known as a translational repressor in vivo is due to its ability to mediate the conformational change of its RNA substrates and thus affect the fate of DDX6-complexed RNA in vivo cross-linking and immunoprecipitation) could help in addressing this question.The biological functions of DDX6 We have also identified two other cellular proteins that affect foamy virus replication; silencing of the poly(A)-binding protein PABPC1 (a stress granule protein) results in a decrease in viral infectivity. PABPC1 is a translation initiation factor but is also involved in many aspects of mRNA metabolism HT1080, 293T, and FAB cells were grown in Dulbecco's modified Eagle's media (DME) supplemented with 10% fetal bovine serum. All virus stocks were derived from an infectious clone of prototype foamy virus, pcPFV Rabbit polyclonal anti-Gag and mouse monoclonal anti-Pol have been described previously gfp gene at the 3′ end of gag, creating a polypeptide with GFP fused at the 631st amino acid of Gag. To generate pcPFV/gag-pum, an oligonucleotide pair of 5′-GACCATCAGATCAAGGTTCTGTAAATACTAGGCCTGTAGATAATCAAGCAGGCTCTGGG-3′ and 5′-CCCAGAGCCTGCTTGATTATCTACAGGCCTAGTATTTACAGAACCTTGATCTGATGGTC-3′ was used to create two adjacent eight-nucleotide sequences (TGTAAATA and TGTAGATA) at the 3′ end of gag. To create BiFC effects, the sequences encoding the split Citrine halves that had been separately fused to either the wild-type or mutant PUMHD domain were cloned into a mammalian expression vector. Thus, pcmv-PUMHD(wt)_CitC and pcmv- CitN_PUMHD(3794) were made by inserting an AflII/XhoI fragment from pGEMTE.PUMHD_CitC or pGEMTE.CitN_PUMHD3794  into an AflII/XhoI digested pcDNA3.1 vector.Proviral construct pcPFV/gag-gfp was generated by inserting a copy of the siRNAs specific to DDX6, Dcp1, PABPC1, Ago1, Ago2, and MOV10, as well as control siRNAs (siCONTROL Non-Targeting siRNA #1 and #5) were from Dharmacon , and some were supplied by Dr. Vineet KewalRamani  . DDX6: 5′-CGAAAUGGCUUAUGCCGCAUU-3′ or 5′-GCAGAAACCCUAUGAGAUU-3′; Dcp1a: pool of 5′-GCAAGCUUGUCGAUAUAUAUU3′, 5′-ACUCAUGGCUGAUGUGGUAUU-3′, 5′-ACAAGCAUCUGACGGUAGAUU-3′, and 5′-CCAAUUCAUUCCUACCAUUUU-3′; PABPC1: pool of 5′-CAUGUAAGGUGGUUUGUGAUU-3′, 5′-GAGCAAGGAAACGUAAUUUUU-3′, 5′-GGACAAAUCCAUUGAUAAUUU-3′, and 5′-UGGAUGAGAUGAACGGAAAUU-3′; MOV10: pool of 5′-CGGCAAGACUGUCACGUUAUU-3′, 5′-GGUCAGAUAUCAGCAAACAUU-3′, 5′-GCCAUGAGGCACAUUGUUAUU-3′, and 5′-CAAUUAAGCAGGUGGUGAAUU-3′; Ago1: pool of 5′-GCACAGUAUUUCAAGCAGAUU-3′, 5′-CAACGAACGGGUCGACUUUUU-3′, 5′-UGACAAGAAUGAGCGAAUUUU-3′, and 5′-GGAAGUACCGCGUGUGUAAUU-3′; Ago2: pool of 5′-GCACGACUGUGGACACGAAUU-3′, 5′-CCAAGGCGGUCCAGGUUCAUU-3′, 5′-GGUCUAAAGGUGGAGAUAAUU-3′, and 5′-CAAGCAGGCCUUCGCACUAUU-3′.5 cells per well in a 6-well plate. The next day, 4 ul of Lipofectamine RNAiMAX  was mixed with 11 ul of sera-free, antibiotics-free DME and incubated for 10 min at RT. This mixture was then combined with 10 ul of the siRNA (either 60 or 120 uM) that had been diluted in 175 ul of sera-free, antibiotics-free DME. After 20 min at RT, the mixture was added to the cells in 0.8 ml of sera-free, antibiotics-free DME. After incubation for 6 hrs at 37°, the complex was removed and replaced with 3 ml of complete media. Cells were transfected with the siRNA again the next day to maximize the knockdown effects.HT1080 cells were seeded at 1×1024 hours after the second siRNA transfection, cells were infected with virus at an moi of 2 for 6 hrs, washed, and replaced with fresh media. Virus titers in culture supernatants were determined by the FAB assay 4 HT1080 cells seeded in a 12-well plate were infected with virus, transfected with 0.7 ug pcPFV or pcPFVgag-gfp, or co-transfected with 0.5 ug pcPFV/gag-pum, 0.1 ug pcmv-PUMHD(wt)_CitC and pcmv- CitN_PUMHD(3794) using Polyfect . Cells were fixed and stained by primary antibodies and Alexa Fluor-conjugated secondary antibodies as described previously 5×10Culture supernatants were filtered and pelleted through 20% sucrose cushion by ultracentrifugation to obtain extracellular particles. The amounts of supernatants to be pelleted were determined according to each sample's intracellular Gag level that was first normalized by GAPDH. The pelleted material was treated with 0.5 mg/ml substilisin  at 37°C for 2 hrs to remove non-specific aggregates, mixed with 0.5 mg/ml PMSF to stop the reaction, and analyzed by Odyssey Western blot.5′-CTACTGGCGCTGCCAAGGCTGT-3′ and 5′-GCCATGAGGTCCACCACCCTGT-3′. Primer sets of 5′-CATAGCGGGACCCGTATAAAAG-3′ and 5′-CAACCAGAGCTTCAACATCAAG-3′ were used to detect unspliced gag RNA.Total cellular RNAs were extracted using RNeasy Mini Kit (QIAGEN). To obtain RNA from the extracellular particles, equal numbers of particles in each sample, as determined by its extracellular Gag level, were treated with RNase-free DNaseI before using QIAamp Viral RNA Mini Kit (QIAGEN). Viral RNA or 1 ug of total cellular RNA from each sample was reverse transcribed using random primers and ThermoScript RT-PCR system (Invitrogen), and subjected to quantitative PCR reactions in triplicates using SYBR Green PCR Core Reagents and ABI 7900HT Real Time PCR Systems . In the quantitative PCR reactions, GAPDH was used as an internal control for cellular RNA with a primer set of 293T cells were transfected with pcPFV for 45 hrs. Cells were washed by ice-cold PBS and incubated for 30 min on ice in a lysis buffer containing 100 mM Tris, pH 7.4, 100 mM NaCl, 10 mM EDTA, 50 mM KAc, 0.625% of NP40, and protein inhibitors. Lysates were centrifuged at 2000 rpm for 10 min followed by another centrifugation at 12,000 rpm for 2 min. The cleared lysates were then incubated with normal rabbit serum, rabbit anti-rck/p54 (referred to as anti-DDX6), anti-hDcp1a, or anti-Gag at 4° for 2 hrs before addition of protein A sepharose . After incubation overnight, the beads were washed 5 times with a wash buffer containing 100 mM Tris, pH 8.0, 100 mM NaCl, and 10 mM EDTA, eluted in 2× SDS-PAGE sample buffer, and analyzed by Immunoblot using mouse anti-Dcp1, mouse anti-DDX6, or mouse anti-Gag.5′-TCGTGTATTTCATGATTTCCGGAACGGTCTCTGTAGCAATCTTGTTTGCACTGATC-3′ and 5′- GATCAGTGCAAACAAGATTGCTACAGAGACCGTTCCGGAAATCATGAAATACACGA-3′) that contains 7 changes in the binding sequences to DDX6 siRNA yet maintains wild type amino acid sequences. 5×104 HT1080 cells per well of a 12-well plate were treated with 120 nM DDX6 siRNA for 24 hrs, transfected with 0.6 ug pcEYFP-C1, or 0.8 ug pEYFP-DDX6a-wt, pEYFP-DDX6a-EQ, or pEYFP-DDX6-dC for 24 hrs, infected with virus at an moi of 2 for 6 hrs, washed, and incubated for another 40 hrs.Expression vector pEYFP-RCK/p54 (referred to as pEYFP-DDX6-wt) was a gift from T. Rana . pEYFP-DDX6-EQ and pEYFP-DDX6-ΔC (referred to as pEYFP-DDX6-dC) were gifts from S. Lemon . pEYFP-DDX6a-wt and pEYFP-DDX6a-EQ were created using primers (


Deficiency of PP4 in B lineage cells leads to a strong reduction in pre-B cell numbers, an absence in immature B cells, and a complete loss of mature B cells. In PP4-deficient pro-B cells, immunoglobulin (Ig) DJH recombination is impaired and Ig µ heavy chain expression is greatly decreased. In addition, PP4-deficient pro-B cells show an increase of DNA double-strand breaks at Ig loci. Consistent with their reduced numbers, residual PP4-deficient pre-B cells accumulate in the G1 phase, exhibit excessive DNA damage, and undergo increased apoptosis. Overexpression of transgenic Ig in PP4-deficient mice rescues the defect in B cell development such that the animals have normal numbers of IgM+ B cells. Our study therefore reveals a novel function for PP4 in pro-B cell development through its promotion of VHDJH recombination.PP4 phosphatase regulates a number of crucial processes but the role of PP4 in B cells has never been reported. We generated B cell-specific  H recombination in Fr. A cells, followed by VH-DJH recombination in Fr. B and Fr. C cells L-JL recombination commences HDJH/VLJL recombination leads to the expression of a surface IgM-containing BCR complex that enables a B cell to continue to the Fr. E and Fr. F stages H/VHDJH recombination is initiated when two Ig gene segments flanked by recombination signal sequences (RSSs) are paired and cleaved by RAG H/VHDJH recombination, an early block in B cell development, and ultimately a shortage of mature B lymphocytes B cell development initiates in the bone marrow (BM) of adult mice and is a tightly controlled process. Developing B cells can be divided chronologically into six Hardy fractions (Frs.) A to F according to the recombination status of the immunoglobulin (Ig) heavy chain (HC) locus, the Ig light chain (LC) locus, and the expression pattern of particular cell surface markers Protein phosphatase 4 (PP4) belongs to the type 2A protein serine/threonine phosphatase (PP2A) family. In mammals, the catalytic subunit of PP4 (PP4c) selectively binds to one or two of several different regulatory subunits, including R1 2+ mobilization and PLC-γ1 phosphorylation normally induced by anti-CD3 stimulation are impaired in these PP4-deficient cells pp4c gene specifically in B cells and identified a pivotal role for PP4 in pro-B cell development. Deletion of PP4 severely disrupted pro-B cell differentiation and consequently led to a complete absence of mature B cells. In PP4-deficient pro-B cells, DJH recombination was greatly reduced and Ig µ HC expression was decreased. We also found that PP4-deficient pre-B cells in vivo accumulated in the G1 phase, showed an elevated level of DNA damage, and underwent increased apoptosis. Significantly, PP4-deficient pro-B cells transgenically expressing IgM successfully differentiated into normal numbers of IgM+ B cells. Our results therefore reveal the indispensable role of PP4 in promoting the VHDJH recombination required for continued pro-B cell differentiation and the production of mature B cells.T cell-specific deletion of PP4 in mice leads to a partial block in thymocyte development at the double negative (DN) stage. The CaF/F mice HEL transgenic mice HEL transgenic and mb-1/cre mice used in all experiments were heterozygous. All protocols were approved by NHRI’s Institutional Animal Care and Use Committee (Permit Number: 099111-A), and all efforts were made to minimize suffering.PP4Cin vivo BrdU incorporation, mice were injected i.p. with 2 mg BrdU (Simga) in 200 µl sterile PBS at 16 h and 24 h before sacrifice and cell sorting. For in vitro BrdU incorporation, isolated cells were cultured for 16 h in RPMI 1640 medium containing 10% FCS (Hyclone) and 10 µM BrdU prior to analysis using a BrdU-Flow kit (BD Pharmingen™).For Single-cell suspensions were prepared from the BM of mice by gentle pipetting and sieving through 70-µm nylon mesh filters . BM cells were cultured in RPMI 1640 medium containing 10% FCS and 2 mM L-glutamine (Invitrogen), 100 U penicillin-streptomycin (Invitrogen), 100 mM Hepes (Sigma-Aldrich), and 0.055 mM 2-mercaptoethanol (Gibco). For flow cytometric analyses, cells were suspended in FACS buffer (2% BSA in PBS containing 0.1% NaN3).To perform comet assays to detect DNA damage, Fr. B and Fr. C cells were sorted by FACSAria (BD) and processed using the OxiSelect™ Comet Assay Kit following the manufacturer’s instructions (Cell Biolabs). Data were analyzed using Comet Assay IV™ v4.3 software.Fr. B and Fr. C cells were sorted, fixed in 4% paraformaldehyde, and permeabilized in 0.2% saponin. After blocking with Image-iT™ FX signal enhancer (Invitrogen), cells were stained with anti-mouse IgM-FITC (HC-specific) antibody (Ab) (BD), seeded on Superfrost® Plus microscope slides (Thermo Scientific), and mounted using Prolong® Gold anti-fade reagent plus DAPI (Invitrogen).H-JH recombination analysis, genomic DNA from 5×105 cells was extracted and subjected to nested PCR utilizing a primer 5′ of the DFL16.1 segment and a primer 3′ of the JH4 segment in the Ig HC locus . To detect DNA double-strand breaks at Ig loci, oligonucleotides BW-1∶5′-GCGGTGACCCGGGAGATCTGAATTC-3′ and BW-2∶5′-GAATTCAGATC-3′ were annealed for the preparation of BW linker as described previously 5′-GCCTTCCACAAGAGGAGAAG-3′ at the upstream of DFL16.1 segment and the primer BW-1  and FACSDiva software (BD) were used for the acquisition of flow cytometric data, and FlowJo software  was used for analysis. For apoptosis assays, cells were stained with B220-APC and Annexin V-FITC using the Apoptosis Detection Kit I (BD). To stain and to sort B cells, BM cells were incubated on ice for 15 min with anti-mouse Abs as follows: BP-1-FITC/PE, IgD-Pacific Blue™ (both from Biolegend); IgM-FITC/eFluor® 450 (eBioscience), B220-PE/PerCP/APC-Cy7, CD21-PE, CD5-PE, CD43-APC, CD23-PE-Cy7, CD24-PE-Cy7, and IgM-APC/PE-Cy7 . Immunostained cells were washed twice in ice-cold FACS buffer prior to sorting by FACSAria cell sorter (BD). For intracellular staining, cells were fixed and permeabilized using the BrdU-Flow kit (BD Pharmingen™) prior to incubation with µ HC-FITC or BrdU-Alexa Fluor® 647 .Single-cell suspensions of 1×10p-values ≤0.05 were considered statistically significant.Data were analyzed by a one-tailed distribution, type 3 Student’s t-test. Differences between treatment groups with pp4c loxp-flanked (floxed) mice F/F (designated here as cKO) mice. As a control, mb-1/cre mice were bred with pp4c WT mice to obtain mb-1/cre;PP4+/+ (designated here as WT) mice. B cell subsets, including Fr. A , Fr. B , Fr. C , Fr. D , Fr. E , and Fr. F , from the BM of WT and cKO mice were analyzed by FACS. In cKO mice, combined staining with anti-B220 and anti-CD43 Abs revealed that the B220+CD43− BM lymphocyte population containing Frs. D to F was greatly reduced (+CD43+ BM lymphocytes on CD24 plus BP-1 expression showed that the percentage of CD24+BP-1+ cells (Fr. C) was strongly reduced in the mutant compared to the control   were present . In parallel to our deletion efficiency determinations, we extracted total RNA from WT and cKO Frs. A-C cells to analyze PP4 mRNA by RT-PCR  at RSSs, the genomic DNA of WT and cKO Fr. B cells was extracted and then subjected to coding end assay as described previously H/VHDJH recombination such that loss of pp4c causes DNA DSBs at Ig loci.During VDJ recombination, the hairpin coding ends are generated after the cleavage of recombination signal sequences (RSSs). Before the rejoining process is initiated, hairpin coding ends are opened to form double-strand ends H/VHDJH recombination by PP4 deficiency raised the possibility that this phenomenon was associated with excessive DNA damage. To address this question, WT and cKO Fr. B and Fr. C cells were subjected to comet assays in which the extent of DNA damage was determined by measuring the percentage of comet tail intensity and the ratio of comet tail length relative to head length. The degree of DNA damage was similar in WT and cKO Fr. B cells  and late apoptotic (AnnexinV+7AAD+) cells were comparable between WT and cKO Fr. B cells, significant increases in early and late apoptotic cells were observed in cKO Fr. C cells compared to WT controls , as was true for the cKO Fr. C culture   . Taken tH/VHDJH recombination. To test this theory, we crossed our cKO mice to IgHEL transgenic mice HEL mice. The IgHEL transgene drives constitutive BCR expression, which should overcome the developmental block in PP4-deficient pro-B cells if this hurdle is caused by impaired Ig DJH/VHDJH recombination HEL mice were analyzed and compared to those of WT and cKO mice. Interestingly, although numbers of Fr. C and Fr. D cells in cKO/IgHEL mice were not restored to WT levels  and IgM+IgD+ cells (Fr. F) in cKO/IgHEL mice had increased to 66.7% and 28.8% . However, the deletion efficiency of the pp4c allele in Fr. A cells was poor, precluding us from investigating the function of PP4 in this population. Fortunately, acceptable deletion efficiencies were achieved in cKO Fr. B and Fr. C cells, permitting us to determine the effects of PP4 deficiency on these subsets. With respect to Fr. B cells, loss of PP4 leads to impaired Ig DJH recombination accompanied by reduced µ HC expression. These defects occur in the absence of abnormal cell-cycle progression or excessive apoptosis. During WT B cell development, secondary DJH rearrangements, which employ a more 5′ D segment to replace the existing DJH rearrangement, occur frequently in pro-B and pre-B cells H/VHDJH recombination, undergo a secondary DJH/VHDJH rearrangement rather than apoptosis. Consistent with that, we found that PP4-deficeint Fr. B cells generate more coding ends at 3′ RSS sites of DFL16.1 and DSP. It suggests that the impaired DJH recombination by PP4 deficiency increases the usage of more 5′ D segments for a secondary rearrangement. Very importantly, the analyses of comet scores and apoptosis in Fr. B cells Click here for additional data file.Figure S2Schematic diagram of southern blotting strategy used to detect coding ends at 3′ of D segments.(TIFF)Click here for additional data file.


V600EB-RAF mutation in 57% of paraffin-embedded biopsies from LCH granuloma. Here we confirm their findings and report the identification of two novel B-RAF mutations detected in LCH patients.Langerhans cell histiocytosis (LCH) features inflammatory granuloma characterised by the presence of CD1a+ dendritic cells or ‘LCH cells’. Badalian-Very et al. recently reported the presence of a canonical V600EB-RAF. In 2 cases novel polymorphisms were identified. A somatic 600DLATB-RAF insertion mimicked the structural and functional consequences of the V600EB-RAF mutant. It destabilized the inactive conformation of the B-RAF kinase and resulted in increased ERK activation in 293 T cells. The 600DLATB-RAF and V600EB-RAF mutations were found enriched in DNA and mRNA from the CD1a+ fraction of granuloma. They were absent from the blood and monocytes of 58 LCH patients, with a lower threshold of sequencing sensitivity of 1%–2% relative mutation abundance. A novel germ line T599AB-RAF mutant allele was detected in one patient, at a relative mutation abundance close to 50% in the LCH granuloma, blood monocytes and lymphocytes. However, T599AB-RAF did not destabilize the inactive conformation of the B-RAF kinase, and did not induce increased ERK phosphorylation or C-RAF transactivation.Mutations of B-RAF were observed in granuloma samples from 11 out of 16 patients using ‘next generation’ pyrosequencing. In 9 cases the mutation identified was V600EB-RAF mutation in LCH granuloma of some patients, and identify two novel B-RAF mutations. They indicate that V600EB-RAF and 600DLATB-RAF mutations are somatic mutants enriched in LCH CD1a+ cells and absent from the patient blood. Further studies are needed to assess the functional consequences of the germ-line T599AB-RAF allele.Our data confirmed presence of the  Langerhans cell histiocytosis (LCH) is a pediatric granulomatous disease with an incidence of four to eight cases per million children + CD4+ T cells LCH lesions feature granulomatous collections of immature CD1a+ langerin/CD207+ DC (‘LCH cells’) presumed to be pathologic V600EB-RAF mutation by pyrosequencing of formalin-fixed, paraffin-embedded material, from 35 out of 61 archived specimens (57%) V600EB-RAF mutation is likely to be somatic, because a germ-line activating V600EB-RAF allele is embryonic lethal in mice Clonality of LCH granulomas has been reported in 1994 V600EB-RAF can contribute to tumour initiation V600EB-RAF induces high levels of cyclin D1-mediated cell proliferation. However, V600EB-RAF also induces oncogene-induced senescence (OIS) that may restrain further development of the tumour V600EB-RAF mutation have been found in a number of benign and malignant tumors including non-malignant naevi V600EB-RAF may represent a first step toward the development of a malignant tumor, although the presence of a V600EB-RAF mutation is not synonymous with cancer.Data obtained from murine model using Cre-mediated activation of a conditionnal B-RAF allele indicate that Of note, the clinical features of LCH are not typical of cancer V600EB-RAF was detectable in the patient's blood, this could be of use for diagnostic, monitoring of treatment efficacy, and potentially prognostic purposes.Identification of dysregulation of the RAS-RAF-MAPK pathway in LCH is nevertheless an important step towards a molecular understanding of the pathophysiology of this pediatric granuloma. It suggests possible novel therapeutic approaches, e.g. the use of B-RAF or MEK inhibitors. Moreover, if We therefore sought firstly to confirm the finding of Badalian-Very using another methodology i.e. analyzing flow sorted CD1a+ cells from fresh LCH granuloma tissue instead of paraffin embedded biopsies. We then aimed to compare relative mutation abundance of V600E B-RAF mutations between CD1a+ and CD1a− cells from LCH granuloma, and whether mutations can be detected in the peripheral blood of patients. Finally we investigated whether additional B-RAF mutations can be found in LCH patients, to further reinforce the link between LCH and the RAS-RAF pathway.V600EB-RAF mutation T599AB-RAF allele , or in data from 1000 genomes project (Nov 10 release).We investigated the presence of B-RAF mutations by ‘next generation’ pyrosequencing (Roche GS FLX 454) in a series of 16 granuloma samples obtained at diagnosis from 16 patients with LCH from a cohort followed in the French LCH Registry  & 2. AmoF allele . The T59V600EB-RAF and 600DLATB-RAF, mutations were detected in granuloma from patients 1–10 with a high relative mutation abundance (RMA)  and in the sample carrying the 600DLATB-RAF mutation (patient #10)  and lymphocytes (CD14−) from patient 1 indicated the absence of detectable B-RAF mutations, with a lower threshold of sequencing sensitivity of 1%–2% relative mutation abundance. Analysis of whole blood from patient 2 with the same method also failed to detect a V600EB-RAF mutation. Blood samples were not available for the 8 other patients with a V600EB-RAF or 600DLATB-RAF mutation.The ce (RMA)  We thereent #10) , CD1a+ cent #10)  were alsent #10) . Genomicent #10) , suggestV600EB-RAF or 600DLATB-RAF were detectable by pyrosequencing in these 56 peripheral blood mononuclear cell samples  , or undeV600EB-RAF and 600DLATB-RAF mutations are detectable in granuloma but not in the blood, and thus strongly suggest they are somatic events. In addition, lack of detection of B-RAF mutations in the blood from patients 1 and 2 and from the 22 others patients for whom blood samples were available at diagnosis or relapse suggests that detection of potential circulating B-RAF mutated cells will require a threshold of sensitivity below 1%.In aggregate, these data indicate that T599AB-RAF mutation was detected in both the granuloma and the whole blood of patient #16. T599AB-RAF was detected in the monocytic (CD14+) and lymphoid fractions (CD14−), at a high frequency of 37 to 45% relative mutation abundance, respectively, similar to its abundance in the granuloma  strongly activates B-RAF 600DLATB-RAF is an in-frame insertion of 12 nucleotides leading to the insertion of 4 amino acids  starting from position 600 .Substitution of Val600 with Glu  strongly activates B-RAF, substitution of Thr599 with Ala (T599AB-RAF) impairs B-RAF kinase activity D594AB-RAF can also transactivate C-RAF and the MEK/ERK pathway, albeit less strongly T599AB-RAF was both impaired in its intrinsic B-RAF kinase activity and unable to transactivate C-RAF. At this time, we do not have a molecular explanation for this observation. Further studies will therefore be needed to potentially unveil the functional consequences of the T599AB-RAF allele on cell activation. The patient's mother born in 1977 carried the same B-RAFT599A allele while in good health, and without a personal history of LCH. Therefore, although the T599AB-RAF allele is not a common polymorphism, there is no evidence that T599AB-RAF is involved in the pathophysiology of LCH in our patient. However the clinical spectrum of LCH is remarkably broad, and the age of onset highly variable While substitution of Val600 with Glu . Primers sets were designed to amplify B-RAF exons 11 and 15 from both genomic DNA and cDNA, which incorporated a universal forward and reverse sequence tag. A second round of PCR was also performed utilizing the universal tags, to incorporate a sample specific 10 bp “barcode" sequence as well as additional tags utilized in the sequencing process (Roche GS FLX Titanium). Sequencing was performed to yield a ‘depth’ in excess of 500 clonal reads (500×) per exonic amplicon, per patient sample in most cases. This allowed detection of mutant clones down to around 1–2% relative mutation abundance, defined as the proportion of sequence reads that contain the mutation. Independent PCR and GS FLX sequencing experiments were carried out to confirm mutations and to reduce sampling error for calculations of mutation abundance. Somatic mutation in patient #16, his parents and his sibling were confirmed using a Applied Biosystem Genetic analyzer 3730xl (primers available on request).600DLATB-RAF and T599AB-RAF have been modeled starting from the structure of the B-RAF kinase domain The B-RAF patient mutations 293 T (Lenti-X 293 T) was purchased from Clontech, and maintained in DMEM supplemented with 10% FBS, 100 units/ml Penicillin, and 100 mg/ml Streptomycin. cDNA fragments of human B-RAF (Genbank accession number: NM_004333) and C-RAF (Genbank accession number: NM_002880) were amplified by RT-PCR from human PBMC cDNA and cloned into pMXs-puro vector. V600E, T599A, D594A, and G596R B-RAF mutants construct were obtained by site-direct mutagenesis (Agilent Technologies). DLAT insertion B-RAF mutant construct was generated by PCR. The constructs were FLAG-tagged and cloned into pMXs-puro and pMXs-IRES-GFP vectors. The mutations were confirmed by DNA sequencing.293 T was transiently transfected with pMXs-puro, FLAG-B-RAF WT, V600E, T599A, 600DLAT, D564A, G596R, or FLAG-CRAFvectors as indicated in the figure. Transfection was carried out with jetPEI according to the manufacture's instructions .Retrovirus packaging cell line, Plat-A was purchased from Cell Biolabs, Inc., and maintained in DMEM supplemented with 10% FBS, 100 units/ml Penicillin, and 100 mg/ml Streptomycin. U937 was kindly donated by Dr. Taams. THP-1 was kindly donated by Dr. Neil. These cell lines were maintained in RPMI supplemented with 10% FBS, 100 units/ml Penicillin, and 100 mg/ml Streptomycin (complete medium). Plat-A was transiently transfected with pMXs-IRES-GFP, FLAG-B-RAF WT, V600E, T599A, 600DLAT, D594A, and G596R vectors using jetPEI. Twenty-four hours after transfection, the medium was changed to fresh DMEM, and the cells were cultured for further 24 hours. The virus supernatants were collected and the debris was removed by centrifuge. The supernatants were mixed with DOTAP Liposomal Transfection Reagent (Roche) and added to U937 or THP-1 culture, followed by centrifuge at 30°C at 1100×g for 2 hours. Forty-eight hours after infection, GFP positive cells were sorted using BD FACSAria (BD Biosciences) and maintained in complete medium.6 of growing cells were colllected by centrifuge and lysed in RIPA buffer. The cell lysates were separated on 10% SDS-PAGE gel and transferred onto nitrocellulose membranes (Bio-Rad). The membrane were blotted with anti-phospho-MEK1/2 mAb (41G9), anti-MEK1/2 mAb (L38C12), anti-phospho-ERK1/2 (D13.14.4E), anti-ERK1/2 mAb (L34F12) all from Cell Signaling Technology, Inc., and anti-FLAG M2 (Sigma Aldrich). Horseradish peroxidase-coupled secondary antibodies were used to detect the primary antibodies. Signal was revealed by enhanced chemiluminescence  using a molecular imager Chemidoc™ XRS+, Biorad. Band intensity was quantified by ImageLab™ Analysis Software.Twenty-four hours after transfection to 293 T cells, culture medium was changed to serum-free DMEM and cells starved for 18 hours. Cells were lysed with RIPA buffer (20 mM Tris-HCl (pH7.4), 150 mM NaCl, 2 mM EDTA, 1% Nonidet-P 40, 0.1% Sodium dodecyl sulfate, 0.1% Sodium deoxycolate, 50 mM Sodium fluoride, 1 mM b-Glycerophosphate, 1 mM Sodium orthovanadate, 1 mM Phenylmethylsulfonyl fluoride, and Protease inhibitor cocktail (Sigma-Aldrich)). For the experiment using THP-1 transfectant, 1×10For the measurement of IL-8 secretion, 1×104 of U937 transfectants ware laid in a 96 well plate in 100 microL. After 24 hours, the plate were centrifuged and supernatants were collected. For pMEK and pERK, 1×104 of U937 were lysed using Bio-Plex Cell Lysis kit. The samples were analyzed according to manufactures instructions.Unsupervised students t-test on single comparisons was compared to analyse significance. A P value<0.05 was considered significant.


In the first step, GFP was engineered to be a foldable, internal methionine-free sequence via the semi-rational mutagenesis of five internal methionine residues and the introduction of mutations for GFP folding enhancement. In the second step, the N-terminus of the engineered protein was modified in vivo with bio-orthogonally functional groups by reassigning functional methionine surrogates such as L-homopropargylglycine and L-azidohomoalanine into the first methionine codon of the engineered internal methionine-free GFP. The N-terminal specific incorporation of unnatural amino acids was confirmed by ESI-MS analysis and the incorporation did not affect significantly the specific activity, refolding rate and folding robustness of the protein. The two proteins which have alkyne or azide groups at their N-termini were conjugated each other by bio-orthogonal Cu(I)-catalyzed click chemistry. The strategy used in this study is expected to facilitate bio-conjugation applications of proteins such as N-terminal specific glycosylation, labeling of fluorescent dyes, and immobilization on solid surfaces.N-terminal site-specific modification of a protein has many advantages over methods targeting internal positions, but it is not easy to install reactive groups onto a protein in an N-terminal specific manner. We here report a strategy to incorporate amino acid analogues specifically in the N-terminus of a protein  It was previously reported that mutations of the three Met residues in the core hydrophobic regions of GFP based on consensus approach induced complete misfolding of the protein We here engineered a green fluorescent protein (GFP) to be an internal Met-free protein sequence and demonstrated its N-terminal functionalization using the Escherichia coli strain M15A methionine auxotroph was kindly donated by Prof. David A. Tirrell  and plasmid pQE80-L was obtained from Qiagen .T4 DNA ligase, restriction endonucleases and PCR reagents, were purchased from New England Biolabs . The isopropyl-D-thiogalactopyranoside (IPTG) and other chemicals were purchased from Sigma chemicals  unless otherwise indicated. Hpg and Aha were purchased from Chiralix . nt. Mutagenesis of the Met codons in GFPnt were carried out by assembly PCR method nt in pQE80-L using two sets of primers  was performed with VentR DNA Polymerase (New England Biolabs) to mutate M153 to Thr. The DNA fragments obtained from these PCR steps were purified on 1.5% agarose gels (Qiagen Gel Extraction Kit). Equimolar quantities of the fragments were used as the template for the assembly PCR using the following pairs of primers: GFPntOut-F and GFPntOut-R. Similarly, M233K mutation was introduced into GFPnt containing M153T using two sets of primers  followed by assembly PCR described above, which generated GFPnt-r2M. GFPnt-r3M was obtained by the saturation mutagenesis of the M218 position in GFPnt-r2M using two sets of primers  and subsequent screening of fluorescent colonies. GFPnt-r4M was also obtained by the saturation mutagenesis of the M78 and M88 positions in GFPnt-r2M using respective sets of primers and subsequent screening of fluorescent colonies. GFPhs-r5M was commercially synthesized by Genescript Corporation . The GFPnt-r2M, GFPnt-r3M, GFPnt-r4M and GFPnt-r5M were cloned into pQE80-L by using BamHI and HindIII restriction sites, and GFPhs-r5M was cloned into the same plasmid by using EcoRI and HindIII sites. All of the constructs possessed hexa-histidine tags in their C-terminals.The pQE-80-GFP constructed in our previous study E. coli M15A Met auxotroph and expressed in minimal medium according to previously described protocols 600 0.6–0.8 and the target proteins were induced with 1 mM IPTG followed by simultaneous addition of either Met or Met analogues (0.5 mM) and allowed the expression for 5 h. The harvested cells were suspended in lysis buffer (50 mM Na-Phosphate buffer pH 7.4 containing protease inhibitor) and disrupted by french press. The suspension was subsequently spun down to collect the soluble and insoluble protein fraction for SDS-PAGE analysis. The remaining soluble protein fractions were purified by Ni-NTA column chromatography  by standard protocol. Elution fractions were analyzed by SDS-PAGE, and those that were enriched in the desired GFP variants were pooled and dialyzed against 1X phosphate buffered saline (PBS).The GFP variants in pQE80-L was transformed into 600, suspended in 1X PBS, by measuring fluorescence intensity by exciting at 485 nm and collecting emission at 515 nm with excitation/emission slits of 5.0 nm using Perkin Elmer/Wallac Victor 2 Multilabel Counter (1420-011). The protein samples were excited at 490 nm and emissions collected at 511 nm with excitation/emission slits of 5.0 nm were recorded on Hitachi FL spectrophotometer (F-4500) equipped with FL solution program for analysis of the spectra.Whole cell fluorescence assay was performed on cells with a 0.1 ODEach purified GFP variant (30 µM) was unfolded at 95°C for 5 min in 1X PBS containing 8 M urea and 5 mM DTT. Refolding was carried out by 100-fold dilution of urea denatured samples at room temperature into 1X PBS containing 5 mM DTT. The concentrations of denatured proteins were adjusted to 0.3 µM and recovered fluorescence was measured using Hitachi FL spectrophotometer  for 30 min with an interval of 3 sec. The recovered fluorescence was normalized by dividing final fluorescence after 24 h value. The normalized values were fitted with Sigma Plot  using equations as described by previous report 0.5 was calculated by measuring the concentration of urea at which the 50% of initial fluorescence was recovered after 24 h of incubation, and the values were determined by sigmoidal fit using sigma plot  Each purified GFP variant (100 µM) was denatured at 95°C for 5 min in 9 M urea in TNG buffer . Equilibrium fluorescence values were measured by diluting the urea denatured proteins into refolding buffer (TNG) containing 5 mM DTT to various final concentration of urea (1–6 M), and allowing the refolding to proceed up to 24 h at 15°C. Fluorescence was measured by exciting at 485 nm and emission at 515 nm with excitation/emission slits of 5.0 nm and was recorded on Perkin Elmer/Wallac Victor 2 Multilabel Counter (1420-011). C4 (50 µl), 50 mM L-ascorbic acid (50 µl), and H2O (300 µl). Control was prepared in similar way without the addition of CuSO4 and L-ascorbic acid. The reaction mixture was shaken for 24 h at 4°C, and dialyzed against 1X PBS thrice at 4°C. Finally, the reaction mixtures were analyzed by SDS-PAGE.Copper (I)-catalyzed cycloaddition reaction between Hpg incorporated GFPhs-r5M and Aha incorporated GFPhs-r5M was performed in a reaction mixture composed of 0.5 mg/ml of each protein (250 µl), 50 mM Tris.HCl pH 8 (100 µl), 50 mM CuSOmut3.1b with a fast folding property nt. Among the five internal Met residues, three Met residues  are located in the hydrophobic core of the protein and the remaining Met residues (M153 and M233) are exposed to solvent. The buried internal Met218 plays a major role in folding process by interacting with single Trp57 through sulfur-aromatic interactions, which is essential for the GFP folding process The wild-type GFP is a 27 kDa protein with a single chain polypeptide containing 238 amino acids. The protein folds into an 11 stranded β-barrel with a single α-helix running through the barrel to form a chromophore. The chromophore cyclization reaction begins once the GFP achieves its near-native like structure nt, designated as GFPnt-r2M, was generated and the effect of mutation on the GFP activity and productivity were examined. GFPnt-r2M showed similar whole cell fluorescence  in GFP was performed. Since the amino acid residues on the protein surface are relatively insensitive to mutations rescence  and solurescence  with GFPnt-r2M in the core hydrophobic region with other amino acids. Actually it was reported that the three Met residues can be mutated into other residues without affecting significantly the fluorescence of cells expressing the GFP variant (GFPrm_AM), in which M78, M88, and M218 were changed into Leu, Phe, and Ala respectively nt-r2M, but the fluorescence of cells expressing the GFP variant  was much lower than that of cells expressing GFPnt-r2M. Since different GFP sequences were used as template to generate GFPrm_AM and the Met-free GFP in this study, we decided to change the three Met residues of GFPnt-r2M step by step. The M218 residue that plays an important role in folding nt-r2M variant having the M218A mutation, designated as GFPnt-r3M, showed the highest fluorescent among the mutants obtained. The whole cell fluorescence of the GFPnt-r3M was approximately 5 times lower than that of GFPnt-r2M . A GFPnt-r2M variant having the M78I and M88L mutations, designated as GFPnt-r4M, showed the highest fluorescence; cells expressing GFPnt-r4M exhibited around 3-fold lower fluorescence than those expressing GFPnt-r2M  of GFPFPnt-r2M . SDS-PAG protein , which hFPnt-r2M . This reFPnt-r2M , and GFPble form . This cont-r5M. It was also reported that N149K nt-r5M, and approximately 2.5 times higher than GFPnt. SDS-PAGE analysis of the expressed protein confirmed that the soluble expression level of the GFPhs-r5M protein was improved significantly compared to that of GFPnt-r5M and higher than that of GFPnt  did not result in an active internal Met-free GFP variant. One thing that needs to be noted is that the starting GFP sequence to generate GFPrm_AM is a GFP variant (L024_3-3) that exhibited higher expression, better refolding behavior and higher stability than normal GFP of GFPnt , suggestin vivo. To demonstrate this, the gene for GFPhs-r5M was expressed in the Met auxotrophic E. coli with the addition of Met surrogates, Hpg or Aha, according to the previously reported procedures The GFPhs-r5M variant obtained from the above study is expressed as a functional form, and contains a Met residue only in its N-terminus, which suggests that the expression of the gene for GFPhs-r5M using the Met residue substitution method may enable the production of N-terminal functionalized GFP nt were also examined and compared as a control.The specific fluorescence, refolding rate and folding robustness of the N-terminal functionalized GFPhs-r5M with Hpg or Aha (designated as GFPhs-r5M-Hpg and GFPhs-r5M-Aha respectively) were compared with those of GFPhs-r5M to examine the biophysical effects of N-terminal functionalization on the protein. The biophysical properties of GFPnt. This indicates that the mutations introduced into GFPnt-r5M for folding enhancement had influence on the spectral properties of protein in addition to the folding efficiency. This also suggests that the higher whole cell fluorescence of GFPhs-r5M than that of GFPnt in As shown in nt, GFPhs-r5M, and GFPhs-r5M with Hpg or Aha. Both GFPhs-r5M-Hpg and GFPhs-r5M-Aha showed similar folding rates in both the fast and slow phases compared to GFPhs-r5M, which were 4–5 fold higher folding rate compared to GFPnt. These results are correlated with the soluble expressions level of GFPnt and GFPhs-r5M  catalyzed cycloaddition reaction of azide and alkyne yields 1,4-disubstituted 1,2,3-triazole linked conjugates under very mild conditions such as room temperature and in an aqueous buffer. Moreover, the reaction is highly regiospecific, chemoselective and tolerant to a wide range of functional groups in vivo. The purified GFPhs-r5M-Hpg and GFPhs-r5M-Aha containing an alkyne and azide groups, respectively, on their N-termini were incubated in the presence of CuSO4 and L-ascorbic acid to carry out the cycloaddition  B. SDS-PAGE analysis of the soluble and insoluble protein fractions of GFPnt-r3M. . C. SDS-PAGE analysis of the soluble and insoluble protein fractions of GFPnt-r5M. . D. SDS-PAGE analysis of the soluble and insoluble protein fractions of GFPhs-r5M and GFPnt. . The expected size of 27.6 kDa is indicated by arrow in the figures.(TIF)Click here for additional data file.Figure S2Chemical structure of natural L-methionine (Met) and their unnatural surrogates L-homopropargylglycine (Hpg) and L-azidohomoalanine (Aha) (Mol. Wt: molecular weight).(TIF)Click here for additional data file.Figure S3ESI-MS analysis of GFPhs-r5M incorporated with Hpg and Aha. GFPhs-r5M (A), GFPhs-r5M-Hpg (B) and GFPhs-r5M-Aha (C) incorporated with Met, Hpg and Aha, respectively.Inset table of each spectra shows calculated and found masses. The peaks corresponding to found masses with Met, Hpg and Aha incorporated proteins might be due to cleavage of 8 residues. We generally could observe these peaks with almost all of the samples of GFPhs-r5M variants. The GFPhs-r5M containing Hpg and Aha showed the mass shift of −22 and −5 Da respectively, compared to GFPhs-r5M with Met.(TIF)Click here for additional data file.Table S1Oligonucleotides used for saturation mutagenesis of internal Met-free GFP construction.(TIF)Click here for additional data file.Table S2Amino acid sequence of the GFPhs-r5M. Red indicates Met replacement mutations, and green indicates the mutations for folding enhancement. The variant expressed as recombinant protein contains a hexahistidine tag sequence in the C-terminus of the protein for Ni-NTA purification.(TIF)Click here for additional data file.


Abnormal accumulation and aggregation of microtubule associated proteins (MAPs) plays an important role in the pathogenesis of neurodegenerative diseases. Loss-of-function mutation of DJ-1/Park7 can cause early onset of PD. DJ-1, a molecular chaperone, can inhibit α-synuclein aggregation. Currently, little is known whether or not loss of function of DJ-1 contributes to abnormal MAPs aggregation in neurodegenerative disorders such as PD.We presented evidence that DJ-1 could bind to microtubule associated protein1b Light Chain (MAP1b-LC). Overexpression of DJ-1 prevented MAP1b-LC aggregation in HEK293t and SH-SY5Y cells while DJ-1 knocking down (KD) enhanced MAP1b-LC aggregation in SH-SY5Y cells. The increase in insoluble MAP1b-LC was also observed in the DJ-1 null mice brain. Moreover, in the DJ-1 KD SH-SY5Y cells, overexpression of MAP1B-LC led to endoplasmic reticulum (ER) stress-induced apoptosis.Our results suggest that DJ-1 acts as a molecular chaperone to inhibit MAP1B aggregation thus leading to neuronal apoptosis. Our study provides a novel insight into the mechanisms that underly the pathogenesis of Parkinson's disease (PD). PD is a common neurodegenerative disease which affects approximately 1% of individuals of 65 years and 5% of those 85 years or older. The featured pathological changes of PD are the selective and progressive loss of dopaminergic (DA) neurons as well as protein aggregation and Lewy body formation . Lewy boIt has been reported that cytoskeletal proteins are involved in the pathology of neurodegenerative diseases ,7. For eLoss of function mutation of Park7/DJ-1 contributed to the pathogenesis of early-onset Parkinsonism . SeveralMAP1b-LC has been shown to be a potential DJ-1 binding protein . To studIt has been reported that MAP1b-LC is a component of cortical Lewy bodies , and abnTo further evaluate the effect of DJ-1 on MAP1b-LC aggregation, MAP1b-LC construct was transfected into HEK293t cells with either DJ-1 or the empty pcDNA3 vector as a control. We observed less MAP1b-LC aggregates in the DJ-1 overexpressed cells compared with the control Figure .The relationship between DJ-1 and MAP1B-LC aggregation was also confirmed in the dopaminergic SH-SY5Y cells. Overexpression of DJ-1 decreased the insoluble MAP1b-LC fraction and inhibited the formation of MAP1b-LC aggregates when they were co-transfected into SH-SY5Y cells Figure . MoreoveL166P, the most common form of DJ-1 mutation, can prevent the dimer formation. The DJ-1 mutant is unstable and is degraded rapidly . These oin vivo. Six-month-old DJ-1 KO mice or wild type littermates were used. The brain lysates of 3 KO or wild type mice were extracted and separated into Triton-X100 soluble and insoluble components. The Western blot results showed an increase in insoluble MAP1b-LC in the DJ-1 KO mouse compared with that of the wild type  and impairments of the UPS in the GAN-null mice may lead to the accumulation of MAP1b-LC . DJ-1 haDown-regulation of DJ-1 can enhance the death of proteasome inhibitor-treated Neuro2A cells , so we aSince protein aggregation is a major cause of neurodegeneration and malfunction of DJ-1 can lead cells to produce more aggregated MAP1b-LC, we next studied whether MAP1b-LC aggregation induced apoptosis of DJ-1 KD SH-SY5Y cells. We transfected the MAP1b-LC into DJ-1 KD SH-SY5Y cells or scrambled control, pDsRed2 being co-transfected with MAP1b-LC for the selection of the transfected cells. After 60 hours, the cells were harvested for the Annexin-V apoptosis assay. We observed more Annexin-V positive cells in the MAP1b-LC transfected group Figure  comparedDJ-1 have been linked to early onset Parkinsonism. However, the molecular mechanism underlying the pathogenesis is still obscure. DJ-1 is thought to be a molecular chaperone and oxidative sensor, participating in both familial and sporadic PD [in vitro and in vivo. Furthermore, aggregates formed by overexpressed MAP1b-LC in HEK293t and SH-SY5Y cells could be inhibited by the overexpression of DJ-1.Mutations of radic PD ,30. Mostradic PD -35 but tradic PD . Our resGAN KO mouse [Protein aggregation and insoluble inclusion bodies are one of the main causes for the pathogenesis of many neurodegenerative diseases . MAP1b iKO mouse . Since DMutations of DJ-1 are thought to be loss function mutations that can lead to autosomal recessive familial PD. The most frequently DJ-1 mutant L166P fails to form dimers and monomeric mutant DJ-1 is unstable and degraded rapidly . TherefoIt has been proposed that the impairments of ubiquitin proteasome system (UPS) play an important role in the pathogenesis of PD. DJ-1 has also been reported to participate in regulating the activity of UPS ,25. It hProtein aggregation has been implicated to play an important role in the pathogenesis of neurodegenerative diseases. Therefore, we analyzed whether excessive MAP1b-LC aggregation can cause cell apoptosis. The data suggested that overexpression of MAP1b-LC in the DJ-1 KD SH-SY5Y cells, which produced overburdened MAP1b-LC aggregates, increased cell apoptosis. It has been reported that abnormal protein aggregation can induce apoptosis mainly through the ER stress pathway . PreviouDJ-1 has been shown to be a molecular chaperone that can inhibit α-synuclein aggregation ,21. Howein vitro and more insoluble MAP1b-LC in vivo. There are two potential explanations for the difference between our in vitro and in vivo experiments: 1) In vitro experiment showed the acute responses of the cells to MAP1b overexpression or DJ-1 KD, in contrast, the in vivo study showed the chronic responses of animals to DJ-1 KO; and 2) both environmental and genetic factors are responsible for the pathogenesis of PD. The absence of MAP1b aggregation in DJ-1 KO mice may be due to the absence of certain non-genetic factors such as aging or neurotoxins in our study.We observed increased aggregation of MAP1b-LC in DJ-1 KD SH-SY5Y cells, as well as an increased level of insoluble MAP1b-LC in DJ-1 KO mice. In contrast, two previous studies did not observe the formation of inclusion bodies in either adult or aged DJ-1 null mice ,39. It sIn summary, we report that DJ-1 is a molecular chaperone that can inhibit the aggregation of MAP1b-LC in vitro as well as the formation of insoluble MAP1b-LC in vivo. Our findings have provided the first evidence that links DJ-1 deficiency to MAPs aggregation, which may improve our understanding regarding the role of DJ-1 in the pathogenesis of PD.The following antibodies were used: DJ-1 Monoclonal Antibody (3E8) , mono- and polyubiquitinylated conjugates, monoclonal Antibody (FK2) , DJ-1 polyclonal antibody , Rabbit polyclonal anti-Flag (F7425), mouse monoclonal anti-Flag (F1804), mouse monoclonal anti-beta-actin (A5441) (Sigma-Aldrich), mouse anti-HA (clone 12C5) , mouse anti-EGFP , Phospho-elF2alpha (Ser51) antibody , Goat polyclonal anti-MAP1b (c-20) . EIF-2α inhibitor Salubrinal was purchased from Calbiochem. All the Chemicals were purchased from Sigma-Aldrich except noted elsewhere.Human DJ-1 and MAP1B-LC cDNA were amplified from the human fetal brain cDNA library (Invitrogen) and ligated to the pcDNA3 vector with an N terminal HA tag and pCMV-3xflag (sigma), respectively. To knock down DJ-1, a DNA fragment and a scramble fragment were synthesized and ligated to the pSilencer-3.1-Hygro (Ambion). The sequence of the inserted DJ-1 DNA fragments is GATCCGCTAAAGGAGCAGAGGAAATTTCAAGAGAATTTCCTCTGCTCCTTTAGTTTTTTGGAAA, and the scramble sequence is GATCCGATCTCTTCTGGTATTAGACTCAAGAGATCTAATACCAGAAGAGATCTTTTTTGGAAA. All the constructions were confirmed by sequencing. PCR-based site directed mutagenesis was used to construct the L166P mutation of DJ-1, which was cloned to the pEGFP-C2 (Clontech).HEK293T and SH-SY5Y cells were purchased from American Type Culture Collection and maintained in DMEM with 10% Fetal Bovine Serum and 100 U/ml penicillin/streptomycin. All the culture materials were purchased from Invitrogen. HEK293t cells were transfected by calcium phosphate precipitation and SH-SY5Y cells were transfected with lipofectamine 2000 (Invitrogen). Transfection efficiency was neutralized by cotransfected with a pRL-tk plasmid. Because DJ-1 L166P is unstable, the amount of the plasmid used for its transfection was 3 times more than that used for the control vector. pSilencer-Hygro-DJ-1 used for DJ-1 KD was transfected into SH-SY5Y and the stable clones were selected with 300ug/ml Hygromycin.For co-immunoprecipitation, HEK293T cells or the mice brain were lysed in the buffer A containing 50 mM Tris·HCl pH 7.6, 150 mM NaCl, 0.5% NP-40, 1% sodium deoxycholate and protease inhibitor cocktail (Roche). To detect the ubiquitination of MAP1b-LC, the cells were harvested and boiled in the lysis buffer B  for 10 min. Then the concentration of SDS in Buffer B was diluted to 0.1% by RIPA buffer. The lysates were pre-cleared with protein A sepharose (GE bioscience) for 30 min. The supernatants were incubated with the primary antibody for 4 hours at 4°C. Then protein A sepharose (GE Bioscience) was added and the mixture was further incubated for 2 h at 4°C. For the Flag fusion protein IP, Flag M2 beads (Sigma-Aldrich) were used. The beads with bound proteins were washed for 6 times with lysis buffer and were boiled in 2X SDS sample buffer, then the samples were detected by immunoblotting.For Western blotting the cells were lysed in RIPA buffer  with protease inhibitor cocktail (Roche). To separate the detergents soluble and insoluble proteins, the homogenized mice brain or the cells were lysed in 0.2% Triton X-100 lysis buffer  on ice for 20 min. Triton X-100-soluble and -insoluble fractions were separated via centrifugation at 13,000 rpm for 15 min [HEK293T or SH-SY5Y cells were grown on glass coverslips, fixed with 4% PFA, permeabilized with 0.2% Triton X-100, and blocked with 20% goat serum or 5%BSA in 0.2% PBST and then incubated with primary antibody. Cells were washed and Alexa 594 or Alexa 488 goat anti-mouse or rabbit IgG antibody (Invitrogen) was added. After washed 3 times using PBS, anti-fade mounting medium with DAPI (Vector Laboratory) was added and the stained cells were analyzed with a confocal microscopy (Leica SP5).Cells were seeded in 6-well plates and transfected with the indicated plasmids and pDsRed2 for 48 hours. Then cells were stained with Annexin V using the Annexin V-FITC apoptosis detection kit (BD bioscience) as the instructions of the manufacturer. Cells  were analyzed using a flow cytometer (Becton Dickinson LSR II).The DJ-1 knock-out mice were kindly provided from Dr. Jie Shen   and crosP value of less than 0.05 and there was no statistical correction was used for all the values.Paired or unpaired Student's t-test was used for statistical analyses. Statistical significance was set at a PD: Parkinson's disease; MAP1b LC: microtubule associated protein 1b; ER: endoplasmic reticulum; KD: Knocking down; KO: Knock out; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; GST: Glutathione-S-transferase; UPS: ubiquitin proteasome system.The authors declare that they have no competing interests.ZQW designed and performed the experiments. YZ contributed to the protein expression and purification. SZ, XR and XYW helped to perform the animal studies. QQG helped to perform the flow cytometry analysis and YYT provided essential advice to the project. SDC and JQD supervised the project and edited the manuscript. All authors read and approved the final manuscript.


Saccharomyces cerevisiae is the preferred microorganism for the fermentative production of ethanol but is not able to ferment pentose sugars. Although D-xylose and L-arabinose fermenting S. cerevisiae strains have been constructed recently, pentose uptake is still a limiting step in mixed sugar fermentations.Hydrolysates of plant biomass used for the production of lignocellulosic biofuels typically contain sugar mixtures consisting mainly of D-glucose and D-xylose, and minor amounts of L-arabinose. The yeast Scheffersomyces stipitis and Stp2 from the plant Arabidopsis thaliana, which mediate the uptake of L-arabinose but not of D-glucose into S. cerevisiae cells. A yeast strain lacking all of its endogenous hexose transporter genes and expressing a bacterial L-arabinose utilization pathway could no longer take up and grow with L-arabinose as the only carbon source. Expression of the heterologous transporters supported uptake and utilization of L-arabinose especially at low L-arabinose concentrations but did not, or only very weakly, support D-glucose uptake and utilization. In contrast, the S. cerevisiae D-galactose transporter, Gal2, mediated uptake of both L-arabinose and D-glucose, especially at high concentrations.Here we described the cloning and characterization of two sugar transporters, AraT from the yeast S. cerevisiae cells, especially at low L-arabinose concentrations.Using a newly developed screening system we have identified two heterologous sugar transporters from a yeast and a plant which can support uptake and utilization of L-arabinose in L-arabinose fermenting  Saccharomyces cerevisiae with yields close to the theoretical maximum [S. cerevisiae is unable to utilize these pentose sugars as fermentative substrates. However, for economically feasible fermentation processes, the bioconversion of all sugars in the raw material is essential.Lignocellulosic biomass represents the most important renewable resource that can be used for the production of biofuels, after its biological conversion into ethanol. D-glucose is the most abundant hexose sugar in lignocellulosic biomass. It can be efficiently fermented to ethanol by the yeast  maximum . D-xylosS. cerevisiae, allowing fermentation of D-xylose and L-arabinose [To overcome this limitation, heterologous pentose utilization pathways from pentose-assimilating organisms have been introduced into rabinose -7. Yet, S. cerevisiae. The hexose transporters of S. cerevisiae, especially Hxt7, Hxt5 and Gal2, catalyze uptake of D-xylose [Interestingly, both pentose sugars, although not metabolized by wild-type yeast strains, can be taken up by D-xylose -10 and GD-xylose . HoweverD-xylose ).S. cerevisiae. In contrast to many bacterial enzymes, heterologously expressed bacterial transporters do not support the uptake of sugars into yeast cells as most of them are not correctly targeted to the plasma membrane . Nevertreported ,13-19. Mreported ,21.Candida spp., Pichia spp., Arxula adeninivorans, Debaryomyces hansenii and Kluyveromyces marxianus have been characterized [Ambrosiozyma monospora were reported to encode specific L-arabinose transporters [S. cerevisiae has not been reported so far. Here, we describe the construction of an L-arabinose transporter screening system based on a S. cerevisiae strain without a hexose/pentose transporter expressing an L-arabinose utilization pathway. This strain is able to grow on L-arabinose media only after functional expression of L-arabinose transporters. Using this screening system, we identified and characterized two transporters from S. stipitis and A. thaliana supporting uptake of and growth with L-arabinose, especially at low L-arabinose concentrations, but not with D-glucose.For L-arabinose uptake, sugar transporters of a few natural L-arabinose metabolizing yeasts like cterized -24. Recesporters . HoweverTAL1 in EBY.VW4000 was exchanged for a strong and constitutive HXT7 promoter fragment [In the yeast strain EBY.VW4000, there are 17 genes encoding all of the members of the hexose transporter family and three genes encoding maltose/glucose transporters which are deleted . As the fragment , resultiS. cerevisiae hexose transporters from Hxt1 to Hxt17 [re expressing the yeast Gal2 galactose transporter and with the empty plasmid p426H7-6HIS as a negative control. Transformants were first plated on selective agar plates without uracil, histidine, tryptophan and leucine, and with maltose as a permissive carbon source. The resulting colonies were streaked out on synthetic medium with 20 g/L L-arabinose as the only carbon source. Transformants expressing ScGal2, ScHxt9 and ScHxt10 could grow on the L-arabinose medium after 10 days of incubation at 30°C, with GAL2-expressing transformants growing the fastest  and low (5 g/L) L-arabinose concentrations, with 20 g/L D-galactose or D-mannose, but only slowly with 20 g/L D-glucose as carbon sources -tag at its C-terminus from plasmid p426-Stp2-HA in strain TSY01. AtStp2 could be identified in Western blots as a distinct band with a size of about 50 kDa , AtStp2, like SsAraT, did not support growth on D-xylose, indicating that AtStp2 is also not able to take up D-xylose (data not shown).For the growth experiments, plasmid pTHStp2 expressing AtStp2 behind the strong fragment  was tranS. cerevisiae cells, growth and sugar consumption were characterized in strain TSY01 expressing the various transporters by shake-flask aerobic batch cultivations. Precultures of the strains expressing the L-arabinose transporters were obtained in SC medium with 20 g/L L-arabinose, while the control strain with the empty vector p426H7-6HIS was pregrown in synthetic complete (SC) medium with 10 g/L maltose. Cells were harvested, washed and inoculated in 50 mL SC medium containing high (20 g/L) or low (5 g/L) L-arabinose concentrations or 20 g/L D-glucose. Growth performance and consumption of sugars were investigated under aerobic conditions  overexpressing SsAraT, AtStp2 or ScGal2 were measured with radioactively labeled sugars Figure . Cells wFor L-arabinose, uptake kinetics were determined by measuring L-arabinose uptake at various concentrations between 0.1 and 50 mM during 2-minute time intervals. While ScGal2 turned out to transport L-arabinose with low affinity and high capacity, SsAraT and AtStp2 mediated uptake of L-arabinose with low capacity but high affinity , supplemented with amino acids but omitting the selective plasmid marker nutrients as described previously [In aerobic batch cultivations, eviously , containS. cerevisiae was grown in SC medium supplemented with maltose, D-glucose or L-arabinose as carbon sources and buffered at pH 6.3 with 20 mM potassium dihydrogen phosphate. Plasmids were amplified in Escherichia coli strain DH5α  or strain SURE . E. coli transformations were performed via electroporation according to the methods of Dower et al. [E. coli was grown on Luria-Bertani medium with 40 μg/mL ampicillin for plasmid selection.For serial dilution growth assays, cells growing in the exponential phase were collected and resuspended in sterile water to an optical density at 600 nm of 1. Cells were serially diluted in 10-fold steps, and 5 μL of each dilution was spotted on agar plates. In aerobic batch cultivations, r et al. . E. coliTAL1 in EBY.VW4000 for the shortened HXT7 promoter was carried out with a modified loxP::kanMX::loxP/Cre recombinase system [loxP::kanMX::loxP-kpHXT7 replacement cassette from the plasmid pUG6-kpHxt7 [The exchange of the endogenous promoter of 6-kpHxt7  was amplGACGGGAAGAGAGA-3') and S2-pTAL2 (5'-CTAGAGAGTTGTTAGCAACCTTTTGTTTCTTTTGAGCTGGTTCAGACATTTTTTGATTAAAATTAAAAAAAC-3') .loxP sites, we routinely used maltose (which has a weaker repressive effect than D-glucose) to induce/derepress loxP-Cre recombination.Yeast transformations were carried out as described previously . As induS. stipitis was obtained from Sloning BioTechnology  by changing the original codons to those used in the highly expressed genes encoding glycolytic enzymes in S. cerevisiae [S. stipitis, the codon of Serin407 was adapted for the usage of S. cerevisiae. The coding region of SsAraT with the optimized codon sequence was amplified and cloned into the vector p426H7-6HIS by recombination cloning [A. thaliana was amplified from pTHStp2 by PCR and cloned by recombination cloning into the vector p426H7-6HIS, fusing a HA-epitope (YPYDVPDYA) at the C-terminal end of AtStp2 but omitting the six histidine codons. Molecular techniques were performed according to published procedures [A synthetic codon-optimized gene version of AraT from revisiae . Becauseocedures .Cultures (50 mL) were grown in 300-mL shake flasks (Erlenmeyer flasks) at 30°C in a shaker. Precultures were grown in SC medium containing 20 g/L L-arabinose or 10 g/L maltose. Cells were washed with sterile water and inoculated to an optical density at 600 nm of 1. All growth assays were carried out at least in duplicate or triplicate.The concentrations of D-glucose and L-arabinose were determined by HPLC (Dionex BioLC) using a Nugleogel Sugar 810 H exchange column . The column was eluted at the temperature of 65°C with 5 mM sulfuric acid as a mobile phase with a flow rate of 0.6 mL/min. Detection was done by means of a Shodex RI-101 refractive-index detector . Chromeleon software 6.50  was used for data evaluation.®), Staufen, Germany). The protein content was determined according to the method of Bradford [Yeast transformants expressing a C-terminally HA epitope-tagged variant of AtStp2 and, as a control, those expressing ScGal2 were cultivated until early exponential growth phase in SC medium with L-arabinose, harvested and disrupted with glass beads 0.45 mm) using a Vibrax cell disrupter -labeled L-arabinose, (U-14C) labeled D-glucose , USA) or (1-14C) labeled D-galactose  was incubated at 30°C and was mixed with 100 μL of yeast suspensions having the same temperature, resulting in final sugar concentrations of 10 mM L-arabinose, D-glucose and D-galactose. For determination of L-arabinose uptake kinetics 0.1, 1, 5, 10 and 50 mM L-arabinose were used. After different time intervals, 10 mL of ice-cold 100 mM potassium phosphate buffer at pH 6.5 with 500 mM D-glucose was added, and the suspension was immediately filtrated using Durapore® membrane filters 0.22 μm pore size , USA). The filter was washed two times with 10 mL of cold potassium phosphate buffer with 500 mM D-glucose. Filters were transferred to 5 mL scintillation vials containing 4.5 mL Rotiszint® eco plus  and the radioactivity measured in a scintillation counter. Uptake of radioactivity was nearly linear in time intervals up to 2 minutes. The results shown are average values for two to three independent experiments. Dry weight was determined by filtering 10 mL of the culture through a pre-weighted nitrocellulose filter . The filters were washed with demineralized water, dried in a microwave oven for 20 minutes at 140 W, and weighted again. KM (Michaelis constant) and vmax  values were calculated using the program GraphPad Prism 5.0 .The initial rates of sugar uptake were measured using a modification of the method described by Bisson and Fraenkel . A 50-μLM : Michaelis constant; vmax: maximal enzyme reaction velocityDM: dry mass; HA: hemagglutinin; HPLC: high perfomance liquid chromatography; kDA: kiloDaltons; PCR: polymerase chain reaction; SC: synthetic complete; KThe authors declare competing financial interests.S. stipitis transporter and the use of the A. thaliana transporter. EB is named as an inventor on both applications. TS is named as an inventor on the application concerning the use of the A. thaliana transporter.Goethe-University Frankfurt has filed two patent applications concerning the use of the TS designed and performed the experiments and wrote the first draft of the manuscript. EB initiated this work, contributed to experimental design and edited the final manuscript. Both authors read and approved the final manuscript.