Saccharomyces cerevisiae, alanine scanning mutagenesis was used to generate point mutants at all possible residues within a GFP-Dbp5 reporter. Characterization of the 456 viable mutants led to the identification of an N-terminal Xpo1-dependent nuclear export signal in Dbp5, in addition to other separation-of-function alleles, which together provide evidence that Dbp5 nuclear shuttling is not essential for mRNP export. Rather, disruptions in Dbp5 nucleocytoplasmic transport result in tRNA export defects, including changes in tRNA shuttling dynamics during recovery from nutrient stress.Dbp5 is an essential DEAD-box protein that mediates nuclear mRNP export. Dbp5 also shuttles between nuclear and cytoplasmic compartments with reported roles in transcription, ribosomal subunit export, and translation; however, the mechanism(s) by which nucleocytoplasmic transport occurs and how Dbp5 specifically contributes to each of these processes remains unclear. Towards understanding the functions and transport of Dbp5 in  In eukaryotic cells, spatial separation of the transcriptional and translational processes necessitates transport of RNA and protein cargos across the nuclear envelope (NE) via nuclear pore complexes (NPCs). Generally, messenger RNAs (mRNAs) are transcribed and processed in the nucleus, transported across the NE, and then translated, stored, or decayed in the cytoplasm. Non-coding RNA (ncRNA) species, such as ribosomal RNA (rRNA) or transfer RNA (tRNA), also undergo nuclear processing and export following transcription . Through6) (DEAD-box proteins (DBPs) are a family of RBPs found in all kingdoms of life, often displaying RNA-stimulated ATPase activity, with diverse roles in RNA biology . For exa6) .Based on the localization of Dbp5 at NPCs and regulation by NPC components, models propose that Dbp5 encounters mRNPs at the cytoplasmic face of NPCs and remodels these substrates to enforce directionality as mRNPs enter the cytoplasm . HoweverXenopus homologs of Dbp5, DDX19B, have been shown to stabilize ribosomal elongation and termination complexes in vitro and contribute to nuclear R-loop clearance upon replication or DNA damage stress , which are part of the namesake D-E-A-D amino acid sequence critical for ATP recognition and hydrolysis. Lethal mutations also mapped to regions of Dbp5 that participate in binding NPC regulators Gle1 and Nup159, including residues R256, Y325, and K382  substitutions at positions 2–482 of Dbp5, resulting in a collection of 481 plasmids carrying s lethal , the majs lethal . For exadrolysis . Similarand K382 . Furtherdbp5-L12A and GFP-dbp5-L15A) showed a prominent nucleoplasmic localization of GFP-Dbp5 -RNA export defect at 37°C, but upon removal of GFP and integration of dbp5-L12A into the genome at the endogenous gene locus, these phenotypes were no longer present , and while the NPC associated pool of Dbp5 was not retained in wild-type during the IF procedure, Dbp5L12A was enriched in the nucleoplasm  were appended to a 2xGFP reporter with an SV40 nuclear localization signal sequence (NLS SV40) to generate a 2xGFP-NLSSV40-NESDBP5 reporter. The addition of NESDBP5 antagonized NLS SV40 activity and facilitated export of the 2xGFP-NLSSV40-NESDBP5 reporter in a manner comparable to a prototypical PKI NES  treatment  sequence  la Cour. To dete PKI NES . Activit in Dbp5 . To testreatment . Upon ad nucleus , which i1 strain . TogetheL12A must fulfill essential activities at NPCs. To test for the possibility that GFP-Dbp5L12A still accesses the cytoplasm, an anchor-away system was used with a peroxisomal anchor, Pex25-FKBP12, to anchor GFP-FRB-Dbp5 (present as sole copy of Dbp5) to peroxisomes in a rapamycin-dependent manner (L12A was rapidly depleted from the nucleoplasm and accumulated on peroxisomes in the same time frame (data not shown). Dbp5 with alanine substitutions at L12, L15, and I17, referred to as GFP-FRB-dbp5ΔNES, also showed similar dynamics -RNA export defect at 37°C, but upon removal of GFP and integration of the mutation into the genome, the dbp5-R423A strain grew comparably to wild-type at all temperatures and had no nuclear poly(A)-RNA accumulation phenotype  to address nuclear access of wild-type or R423A versions of GFP-FRB-Dbp5 when present as sole copy. Upon addition of rapamycin, GFP-FRB-Dbp5 moved to the nucleoplasm, resulting in complete depletion of the GFP signal at the NE within ~11 minutes . In both wild-type cells and dbp5-R423A strains, GFP-FRB-Dbp5 rapidly accumulated on peroxisomes after rapamycin addition, resulting in complete depletion of the GFP signal from the NE in both strains in ~4 minutes (R423A nuclear import defect (data not shown), which is suggestive of competition for nuclear transport. These results show that GFP-FRB-Dbp5R423A remains dynamic in the cytoplasm, but Dbp5R423A import efficiency is altered by this mutation.To assay Dbp5 minutes . GFP-FRBg series . To test minutes . Note th minutes . A majordbp5-R423A allele and discovery of an N-terminal NES within Dbp5 provides a means to investigate the mechanics and functional importance of Dbp5 nuclear shuttling. Markedly, the lack of poly(A)-RNA accumulation in integrated versions of dbp5-L12A, where Dbp5 is predominantly nucleoplasmic, or dbp5-R423A, where Dbp5 nuclear access is altered, suggests that neither a large cytoplasmic pool of Dbp5 or rapid shuttling of Dbp5 through the nucleus is required to maintain steady-state nuclear mRNP export.Overall, the altered import kinetics of the dbp5-L12A) or altering nuclear access (i.e. dbp5-R423A), a synthetic gene array (SGA) screen was carried out to identify potential genetic interactions between these alleles and non-essential gene mutants. SGA screens have been carried out previously with the DBP5 Ts allele rat8-2, yielding 108 potential synthetic interactions, encompassing proteins involved in chromatin remodeling, transcription, mRNA metabolism, and mRNP export .Recent work has linked Dbp5 to the export of ncRNAs, including ribosomal and telomerase RNAs . Here, SIleUAU in strains carrying integrated and untagged versions of wild-type, dbp5-L12A, and dbp5-R423A. The probe spans the 5´ exon and first 30 nucleotides of the tRNAIleUAU intron to allow detection of the primary transcript and processing intermediates. The ratio of intron containing intermediate (I) to precursor transcript (P) normalized to wild-type is used as a measure of tRNA processing, with altered ratios indicating a defect (dbp5-L12A (1.00 ± 0.1) was unchanged from wild-type, while a small but consistent accumulation of the intron-containing tRNA intermediate was observed in dbp5-R423A (1.4 ± 0.1) (dbp5-R423A (2.3 ± 0.2), while the ratio only increased slightly in dbp5-L12A (1.4 ± 0.2)  at 37°C  . This de4 ± 0.2) . To veri at 37°C . These mdbp5-R423A by northern blotting are suggestive of a defect in tRNA export , accumulation of the intron containing tRNA intermediate was similar in the untagged and integrated dbp5-L12A/R423A (1.9 ± 0.5), suggesting that depleting the cytoplasmic pool of Dbp5 through inclusion of the L12A mutation did not further impact tRNA processing, in contrast to what is seen with mRNP export. Due to the presence of mRNP export defects at 25°C, this mutant was not used to further interrogate the role of Dbp5 in tRNA export. Combined, the SGA, northern, and in situ data employing the dbp5-L12A and dbp5-R423A mutations to alter Dbp5 nuclear levels are strongly supportive of Dbp5 functioning in tRNA export within the nucleus, while facilitating mRNP export in the cytoplasmic compartment (e.g. at the cytoplasmic face of NPCs).Dbp5to R423A ; however at 37°C . This re at 37°C . As compnup159-1  leveraging the strong nuclear signal of GFP-Dbp5L12A to assess Dbp5 nuclear localization. Under these conditions, it was observed that nuclear GFP-Dbp5L12A localization was only reduced in gle1-4 at 37°C, but remained largely unchanged in gle1-4 at 25°C or nup159-1 and mex67-5 at 25°C or 37°C  strains; however, in dbp5-R423A the majority of cells at 10 minutes still showed elevated levels of nuclear tRNA (74 ± 9). These observations, plus the genetic interaction with a msn5Δ, support the conclusion that nuclear Dbp5 supports the re-export of mature tRNAs following stress. The smaller but significant perturbation in the re-export of tRNAs in dbp5-L12A also suggests that efficient nucleocytoplasmic shuttling mediated by the N-terminal NES is important in the context of tRNA re-export.It is known that re-export of mature tRNAs to the cytoplasm in response to nutrient availability involves the β-importin family member Exportin-5 (yeast Msn5) . The msn  controls  tagged Dbp5 followed by RT-qPCR analyses were performed to determine if Dbp5 forms a complex with tRNAs. Using this approach, PrA-Dbp5-RNA interactions were enriched for unspliced and spliced versions of tRNAl strain . PrA-Dbp05 level . In contNAIleUAU . These pcontrols . OverallDED1/DDX3 homologs and DDX28 -RNA accumulation phenotype at 37°C for both GFP-dbp5-L12A allele had no growth defect at 25°C or 37°C, no mRNP export defect detectable by FISH, and minimal genetic interactions as measured by SGA. Similarly, dbp5-R423A showed no growth defects or detectable poly(A)-RNA accumulation at any temperature. Together these data support the general conclusion that Dbp5 nuclear shuttling is not essential to Dbp5 mRNP export.The inclusion of GFP in this collection and subsequent identification of specific mutants that impact nuclear shuttling of Dbp5 was critical to establishing a nuclear function of Dbp5 reported here. This was accomplished through screening the mutant collection in the presence and absence of ethanol stress, which led to the identification of the Xpo1 dependent N-terminal NES sequence in Dbp5  and a stImportantly, this work further provides information that can be used to discern current models describing the function of Dbp5 during mRNP export (reviewed in TLC1 (DBP5 shuttling mutants (dbp5-L12A or dbp5-R423A) were not observed under the conditions tested. This may be due in part to multiple redundant pathways that contribute to pre-ribosomal subunit export, which includes Arx1, and the essential non-karyopherin transport receptor required for mRNP export, Mex67 (TAP or NXF1)  . The lacdbp5-R423A strain after a temperature shift to 37°C or following addition of nutrients (e.g. amino acids) to starved cells. Re-introduction of nutrients is a condition that induces re-export of a large nuclear pool of mature tRNAs that accumulate during nutrient starvation  of Dbp5, and how this activity is modulated in vivo, to support mRNA, tRNA, and rRNA export. For example, does Dbp5 generally function to remodel all RNP complexes exiting NPCs or does Dbp5 function in a distinct manner on mRNA, tRNA, and rRNA substrates (e.g. RNPase vs. scaffold models)? For mRNAs, data presented here supports the conclusion that Dbp5 functions at NPCs during mRNP export, in line with an RNPase model . In contFor a list of DBP5 mutagenesis primers, yeast strains, plasmids, qPCR primers, FISH probes, and Northern probes see E. coli cells and plated onto LB ampicillin in a 12 well plate format. Mini preps were performed in a 96 well format (BioBasic Inc) on selected single colonies to purify mutagenized plasmids. The presence of each mutation was verified by sequencing (Genome Quebec).A plasmid (pBM464) containing DBP5 ±500 bp of flanking sequence was generated with an N-terminal GFP sequence fused to the DBP5 coding region as a template for mutagenesis. Primers for alanine scanning mutagenesis were designed using the AAscan program against all residues . PCR wasdbp5Δ strain carrying DBP5 on a URA3 marked CEN plasmid (BMY015). The BMY015 strain was grown to an O.D600 of 1.0 in 1.5 L of selective media, centrifuged at 3000xg for 5 min, washed once in water, and then in 50 ml of 100 mM lithium acetate (LiAc). The culture was re-suspended in 30 ml of 100 mM LiAc and 50 μl of culture was aliquoted into each well of a 96-well plate. Plates were centrifuged at 3000xg for 5 min and supernatant was removed. Cells were re-suspended in a transformation mix . Mutated plasmid DNA (250 ng) was added to each well and mixed. Plates were incubated at 30°C for 45 min and then at 42°C for 30 min. Plates were centrifuged again and the supernatant was removed and 150 μl of synthetic complete (SC)-LEU/-URA media was added. Cultures were grown for 3 days at 25°C and then 5 μl of culture was diluted into 200 μl of fresh media and grown for another 2 days. Transformants were then pinned for two rounds onto SC/-LEU/-URA selective agar plates and grown at 25°C. To remove the wild-type plasmid, strains were also pinned on to 5-FOA for two rounds of selection before finally pinning onto SC/-LEU plates. Strains that did not grow following plating on 5-FOA were considered to contain lethal mutations. To determine which mutant strains were temperature sensitive, the mutant array was plated on SC –LEU plate, grown at 37°C for 3 days, and then repined on SC –LEU and grown at 37°C for 3 days. Strains that did not grow when compared to a 25°C control plate were noted to be temperature sensitive and were subsequently verified by additional spot plating assays at 37°C.Sequence verified plasmids were transformed into yeast in a 96-well plate format using a Live cell imaging was performed using a widefield or confocal configuration on an Andor Dragonfly microscope equipped with an EMCCD camera driven by Fusion software (Andor) with a 60x oil immersion objective . For imaging of the mutant collection, strains were grown overnight in 96 well plates at 25°C to mid-log phase in SC media and then placed in 384-well glass bottomed plates (VWR) treated with Concanavalin A. To determine changes in localization as a result of ethanol stress, media containing 24% ethanol was added to each plate well to achieve a final concentration of 12% ethanol, followed by imaging 15 min after ethanol addition. Anchor away experiments were similarly performed in glass bottom plates using 1 μg/ml rapamycin, pre-treated with or without 100 ng/ml leptomycin B, followed by imaging for 20 min at 1 min intervals. To quantify the nuclear/cytoplasmic fluorescence intensity of GFP-NLS/NES reporters, the mean integrated fluorescence intensity was measured for a 0.5 μm x 0.5 μm region in the nucleus or cytoplasm. Signal intensity was normalized to a background fluorescent intensity value adjacent to each cell and used to calculate the nuclear/cytoplasmic ratios. For each condition, measurements for 100 cells were obtained and the average ratios were plotted in a bar graph with the variability between cells expressed as standard deviation. Image analyses were performed in FIJI, including maximum z-projections, image cropping, and brightness/contrast adjustments .SDS-PAGE was carried out as described above. Gels were fixed with 50% ethanol 2x for 15 min followed by 20 min incubation in DTT (5 μg/ml) and a 20 min incubation in 0.1% AgNO3. Finally, the gel was developed in developing solution (3% Na2CO3 (w/v), 2% formaldehyde) and stopped by incubation in 1% Acetic Acid.2). To spheroplast cells, 2.5 µL 20 mg/mL zymolyase T20 and 5 µL of 200 mM vanadyl ribonucleoside complex (VRC) were added to cells re-suspended in 425 µL of Buffer B for 30 min at 30°C. Samples were washed once with Buffer B and then re-suspended in 70% ethanol and incubated for 4 hr at room temperature. After washing samples with Buffer B, hybridization was carried out in 50 ul of buffer containing 1X saline sodium citrate (SSC), 0.34 mg/ml E. coli tRNA, 20% formamide, 0.2 mg/ml BSA, 11% dextran sulphate, 4 mM VRC, and oligo-dT or gene specific probes as indicated at 37°C for 16 hr. A final concentration of 0.4 mM was used for the fluorescein isothiocyanate (FITC) labeled oligo dT probe (Qiagen). Samples were washed two times with 1X SSC and 15% formamide followed by application onto 8-well slides (Fisher Scientific) treated with Poly-L-Lysine (SIGMA). After washing twice with 1x phosphate buffered saline (PBS), mounting medium with DAPI was applied to each sample and a coverslip was affixed. For tRNA specific FISH was carried out as previously described by 600=0.3) in YPD at 25°C  followed by formaldehyde fixation at a final concentration of 3.7% for 15 min. Samples were then re-suspended in 5 ml of fresh Buffer A  for 3 hr at room temperature. Cells were then washed two times with Buffer B. To spheroplast cells, 8 µL of 20 mg/mL zymolyase T20 and 1 µL of β-mercaptoethanol was added to cells re-suspended in 500 µL of Buffer B for 30 min at 37°C. Cells were then washed one time with Buffer B and adhered to eight well slides coated with Poly-L-lysine. Slides were treated with 70%, 90%, and 100% ethanol for 5 min. Cells were then incubated in a pre-hybridization buffer , 2 × SSC (1 × SSC is 0.15 M NaCl and 0.015 M Na-citrate), 1X Denhardt’s solution, 250 μg Escherichia coli tRNA/ml) for 2 hr at 37°C. For hybridization, 1 pmol/ul of Cy3-dUTP end labeled tRNA probes were added to fresh hybridization buffer and incubated overnight at 37°C. All tRNA FISH probes were 3’end labeled using an oligonucleotide end labeling kit (Roche) and Cy3-dUTP (Enzo Life Sciences). Cells were washed one time in 2xSSC at 50°C, and then two subsequent washes for 10 min at room temperature each in 1X SSC, 4X SSC, 4X SSC +1% Triton X-100. Slides were then washed in 4X SSC again briefly, and once in 1X PBS and then mounted with DAPi and sealed. Imaging was performed using a microscope  equipped with an EMCCD camera driven by Fusion (Andor) using a 60x × 1.4 NA oil objective (Olympus). Image analysis was performed in FIJI, including maximum z-projections, image cropping, and brightness/contrast adjustments (FISH was carried out as described with modifications . Brieflyustments .2) and re-suspended in 1 ml of Buffer B . To spheroplast cells, 2.5 µL 20 mg/mL zymolyase T20 and 1 µL of β-mercaptoethanol were added to cells for 30 min at 37°C. Samples were washed once with Buffer B and then adhered to eight well slides coated with Poly-L-lysine. Slides were treated ice-cold methanol for 6 min and then acetone for 30 s. Samples were blocked with 1X phosphate buffered saline with 5 mg/ml bovine serum albumin (PBS-BSA) for 1 hr at room temperature. Samples were incubated with a mouse monoclonal anti-DBP5 primary antibody overnight at room temperature in PBS-BSA with 0.05% Tween-20. Slides were then washed five times with PBS-BSA-Tween for 5 min each and then incubated with a Goat anti-mouse DyLight 650 (Thermo Fisher) secondary antibody in PBS-BSA-Tween. Slides were then washed five times with PBS-BSA-Tween for 5 min each, and twice in 1X PBS and then mounted with DAPi and sealed. Imaging was performed using a microscope  equipped with an EMCCD camera driven by Fusion (Andor) using a 60x × 1.4 NA oil objective (Olympus). Image analysis was performed in FIJI, including maximum z-projections, image cropping, and brightness/contrast adjustments . Membranes were cross-linked at 2400 J/m2 . tRNAs were detected using with digoxigenin-labeled (DIG) probes as described previously , Protease Inhibitor Cocktail was added to 1X concentration and lysis was performed by vortexing with 1 ml Zirkonia beads (0.5 mm) 5 times for 1 min with 1 min incubation on ice between each cycle. 5 ml TN150 was added and lysate was cleared by centrifugation . Lysates were diluted to 10 ml with TN150 and incubated with IgG-conjugated magnetic dynabeads at 4°C 1 hr with rotation. Immuno-precipitate was washed once for 5 min with 1 ml TN150, followed by 5 min 1 ml TN1000 , and once more with 1 ml TN150 for 5 min at 4°C with rotating. Immuno-precipitate was re-suspended in 100 μl TurboDNase reaction  and incubated on a thermomixer at 37°C for 30 min at 1200 RPM. Magnetic beads were washed 3 times with 1 ml TN150 and 50 μl of beads were retained for western blot analysis. Remaining beads were aspirated of TN150 and re-suspended in 400 μl Proteinase K elution mix spiked with exogenous Luciferase RNA . Elution was carried out at 50°C 1200 rpm for 2 hr on thermomixer. Magnetic beads were incubated at 65°C for 1 hr to allow cross-link reversal by heat. RNA was extracted from IP with 400 μl Sigma 5:1 phenol:chloroform pH 4.3–4.7 followed by back extraction with chloroform:isoamylalcohol and ethanol precipitation for 1 hr at −80°C using linear acrylamide as a carrier. Half volume of RIP RNA was reverse transcribed with Superscript III using random priming, while the other half was retained for -RT reaction to assess genomic contamination. Reverse transcription was carried out as per manufacturer’s instructions. Resulting cDNA from RIPs was used in standard PCR followed by 2% gel electrophoresis in addition to being used as template in qPCR to quantify fold enrichment of target RNAs in RIP.RT-qPCR experiments were carried out using Power SYBR (Applied Biosystems) on an Applied Biosystems instrument. Target RNA abundance in each RIP was normalized to the abundance of an exogenous spike-in Luciferase RNA control to correct for differences in sample preparation (dCt). ddCt values were then calculated as the difference in relative abundance of the target RNA isolated via immunoprecipitation using a Protein-A tagged Dbp5 strain vs. an untagged strain (non-specific binding control). Relative fold-enrichment above the untagged background control was calculated using the Livak Method . Standar In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.eLife. Your article has been reviewed by Kevin Struhl as the Senior Editor, a Reviewing Editor, and two reviewers, a Reviewing Editor, and two reviewers. The reviewers have opted to remain anonymous.Thank you for submitting your article "A nuclear role for the DEAD-box protein Dbp5 in tRNA export" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.The authors conduct a comprehensive analysis of the roles of the budding yeast DEAD box protein, Dbp5, in RNA nuclear export, RNA response to environmental stress, and protein synthesis. The studies are based upon an alanine scanning mutant collection they created and their extensive analyses including: growth phenotypes, live cell imaging of the localization mutant tagged Dbp5 proteins, protein localization motifs via reporter constructs, Sup45 co-IP, subcellular protein tethering, RNA FISH localization of poly A mRNA and tRNA, tRNA co-purification with Dbp5; RNA seq changes in response to stress, and SGA for two dbp5 alleles!eLife a significant re-writing is suggested. The main point of the manuscript, a role of Dbp5 in tRNA metabolism, is appropriately supported. Moreover, the different roles of Dbp5 in mRNA nuclear export vs. tRNA nuclear export are interesting, including: the different motifs important for both, the data indicating that nucleus-cytoplasm Dbp5 shuttling seems not to be important for mRNA nuclear export (supporting the model of Dbp5 functioning to release mRNA on the cytoplasmic surface), whereas it appears to be important for tRNA nuclear export (support a model of Dbp5 assembling tRNA in an export complex).Due to the massive data sets and analyses the manuscript is quite exhausting to read and the reviewers fear that it will be to disperse and out of reach for non-experts. Still, the authors report results of scientific interest and validity. Hence, to make the paper scientifically appropriate for In contrast, the reviewers believe, that the sections describing the RNA-seq analyses, the translation inhibitor studies and MMS screen do not make a coherent scientific point, but could potentially be assembled into separate manuscripts.1) Tailoring of the manuscript throughout would really help its impact. Models/cartoons – diagrams often convey messages more effectively than text. For example, a cartoon indicating how Dbp5 may function differently in mRNA vs. tRNA nuclear export could help readers understand the complexity of Dbp5's biological roles and the roles of different motifs in the various functions.2) Figure labeling – it would be easier to access the data if the labeling of figures were improved. For example: Figure 2—figure supplement 1B and C appear similar, but B is FISH and C is protein imaging; so, putting this information on the figure in addition to the legend would be helpful to the readers. Another example for Figure 5; it would be helpful to label A. tRNAIle, B. tRNATyr, C. FISH tRNATyr, D. tRNAIle. Also, it is not clear in the figure or the text that A and D were conducted in Dbp5-tagged vs. untagged strains.3) tRNA experiments: Figure 5C and D; why is the FISH conducted for tRNATyr whereas the northern for tRNAIle? Figure 6, even though L12A doesn't have a very remarkable affect upon tRNA nuclear accumulation, its response to -AA likely should be included in Figure 6. tRNA RIP analyses should include an internal nonspecific RNA, such as a mitochondrial tRNA, to assess pull-down specificity. One would have expected that L12A and R423A would have shown different levels of tRNA pulldown given their different roles as analyzed by FISH; how do the authors think about this.dbp5-L12A and dbp5-R423A at steady-state and in response to MMS”: no data are shown for the claim that steady state RNA levels were similar for wt, L12A and R423A mutants.4) Subsection “Gene expression in 5) Figure 4 – it is nearly impossible to read the identities of the genes through the magenta and purple colors.6) Figure 4A,B: differences in RNA levels in the mutants upon MMS treatment would be better shown as correlation plots. The data are hard to parse in their current form.dbp5-L12A and dbp5-R423A at steady-state and in response to MMS”: The FDR values for the highlighted GO terms are large (i.e. barely significant), raising the question whether the reported enrichments are biological meaningful. The authors make the point that L12A and R423A cause opposite changes in gene expression in the GO categories. Are the same genes affected? If so, this should be shown. If not, the FDR values for enrichment would probably render this effect statistically insignificant. More depth is needed in this section.7) Subsection “Gene expression in dbp5-L12A and dbp5-R423A genetic interaction profiles”: The referenced Figure 4 – figure supplement 1 seems to be missing.8) Subsection “eLife. Your revised article has been favorably evaluated by Kevin Struhl (Senior Editor), a Reviewing Editor, and two reviewers.Thank you for resubmitting your work entitled "A nuclear role for the DEAD-box protein Dbp5 in tRNA export" for further consideration at The manuscript has been improved and acceptance is recommended in principle, but there is one remaining issue that needs to be addressed before acceptance, The new Figure 6 does not provide identification for all the proteins, either on the figure or in the legend. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.The authors conduct a comprehensive analysis of the roles of the budding yeast DEAD box protein, Dbp5, in RNA nuclear export, RNA response to environmental stress, and protein synthesis. The studies are based upon an alanine scanning mutant collection they created and their extensive analyses including: growth phenotypes, live cell imaging of the localization mutant tagged Dbp5 proteins, protein localization motifs via reporter constructs, co-IPs, subcellular protein tethering, RNA FISH localization of poly A mRNA and tRNA, tRNA co-purification with Dbp5; RNA seq changes in response to stress, and SGA for two dbp5 alleles!We wish to thank the reviewers and editors for their constructive and helpful comments. Please find below our response to each.Due to the massive data sets and analyses the manuscript is quite exhausting to read and the reviewers fear that it will be to disperse and out of reach for non-experts. Still, the authors report results of scientific interest and validity. Hence, to make the paper scientifically appropriate for eLife a significant re-writing is suggested. The main point of the manuscript, a role of Dbp5 in tRNA metabolism, is appropriately supported. Moreover, the different roles of Dbp5 in mRNA nuclear export vs. tRNA nuclear export are interesting, including: the different motifs important for both, the data indicating that nucleus-cytoplasm Dbp5 shuttling seems not to be important for mRNA nuclear export (supporting the model of Dbp5 functioning to release mRNA on the cytoplasmic surface), whereas it appears to be important for tRNA nuclear export (support a model of Dbp5 assembling tRNA in an export complex).In contrast, the reviewers believe, that the sections describing the RNA-seq analyses, the translation inhibitor studies and MMS screen do not make a coherent scientific point, but could potentially be assembled into separate manuscripts.The manuscript has been rewritten to focus on Dbp5 nuclear transport and the role of Dbp5 in tRNA metabolism. This includes removal of the phenotypic screens and RNA-seq data as suggested. We believe this has improved overall of clarity and more effectively conveys the impact of the work. Again, we thank the reviewers for this suggestion.1) Tailoring of the manuscript throughout would really help its impact. Models/cartoons – diagrams often convey messages more effectively than text. For example, a cartoon indicating how Dbp5 may function differently in mRNA vs. tRNA nuclear export could help readers understand the complexity of Dbp5's biological roles and the roles of different motifs in the various functions.As noted above, rewriting has aided with both message and impact. We have also added a model figure to help convey potential ways Dbp5 is functioning in tRNA vs. mRNA export .2) Figure labeling – it would be easier to access the data if the labeling of figures were improved. For example: Figure 2—figure supplement 1B and C appear similar, but B is FISH and C is protein imaging; so, putting this info. on the figure in addition to the legend would be helpful to the readers. Another example for Figure 5; it would be helpful to label A. tRNAIle, B. tRNATyr, C. FISH tRNATyr, D. tRNAIle. Also, it is not clear in the figure or the text that A and D were conducted in Dbp5-tagged vs. untagged strains.We have added labels as requested, plus reviewed all figures and legends to improve labeling and clarity.3) tRNA experiments: Figure 5C and D; why is the FISH conducted for tRNATyr whereas the northern for tRNAIle?Figure 5C/D, now Figure 4C/D, were both performed with tRNAIle, which was not apparent based on figure labeling. Related to comment #2 above, we have now added labels to resolve this issue.Figure 6, even though L12A doesn't have a very remarkable affect upon tRNA nuclear accumulation, its response to -AA likely should be included in Figure 6.We have added L12A data as requested. Please see current Figure 5 .tRNA RIP analyses should include an internal nonspecific RNA, such as a mitochondrial tRNA, to assess pull-down specificity.We have now assessed pulldown specificity by looking for the mitochondrial COX1 mRNA in cDNA libraries generated from all RIP experiments used to generate the data presented in Figure 5B. The COX1 transcript was not detected in any of the Dbp5 RIPs at levels above the -RT background; however, it was present in cDNA libraries made from total RNA. These data are discussed in the text in subsection “Dbp5 supports re-export of mature tRNAs following nutritional stress” and shown in Figure 5—figure supplement 1C.One would have expected that L12A and R423A would have shown different levels of tRNA pulldown given their different roles as analyzed by FISH; how do the authors think about this.As shown in Figure 5 and discussed in subsection “Dbp5 supports re-export of mature tRNAs following nutritional stress” and subsection “Dbp5 acts within the nucleus to support tRNA export” of the revised manuscript, L12A has a significant enrichment for binding tRNAIle. We interpret this increased interaction to be reflective of the increased nuclear pool of Dbp5. In the case of R423A, the interaction with unspliced tRNAs appeared reduced potentially corresponding with altered nuclear import, but this did not reach a significance level of p<0.05. Still, reduced binding is not the only means by which this mutation could impact tRNA export. For example, our data shows that RNA does not stimulate the ATPase activity of R423A to the level of wildtype Dbp5. As such, although binding events occur and are detected, binding may not be coupled with productive ATPase cycles that are required to support tRNA export. We have expanded the discussion of the R423A mutant in subsection “Dbp5 acts within the nucleus to support tRNA export” of the revised manuscript to reflect these comments.4) Subsection “Gene expression in dbp5-L12A and dbp5-R423A at steady-state and in response to MMS”: no data are shown for the claim that steady state RNA levels were similar for wt, L12A and R423A mutants.Through revision of the manuscript this statement has been removed.5) Figure 4: it is nearly impossible to read the identities of the genes through the magenta and purple colors.Colors have been updated to allow gene names to be more easily read. Please see current Figure 3 .6) Figure 4A,B: differences in RNA levels in the mutants upon MMS treatment would be better shown as correlation plots. The data are hard to parse in their current form.Through revision of the manuscript these data have been removed.7) Subsection “Gene expression in dbp5-L12A and dbp5-R423A at steady-state and in response to MMS”: The FDR values for the highlighted GO terms are large (i.e. barely significant), raising the question whether the reported enrichments are biological meaningful. The authors make the point that L12A and R423A cause opposite changes in gene expression in the GO categories. Are the same genes affected? If so, this should be shown. If not, the FDR values for enrichment would probably render this effect statistically insignificant. More depth is needed in this section.Through revision of the manuscript these data have been removed.8) Subsection “dbp5-L12A and dbp5-R423A genetic interaction profiles”: The referenced Figure 4—figure supplement 1 seems to be missing.Through revision of the manuscript these data have been removed.The manuscript has been improved and acceptance is recommended in principle, but there is one remaining issue that needs to be addressed before acceptance, the new Figure 6 does not provide identification for all the proteins, either on the figure or in the legend.A nuclear role for the DEAD-box protein Dbp5 in tRNA export” as requested. Specifically, we have added greater detail to the model presented in Figure 6 and the associated legend describing the figure.We have revised the manuscript by Lari et al., entitled “


Sci., 2015, 6, 3775–3787.Correction for ‘Supramolecularly engineered phospholipids constructed by nucleobase molecular recognition: upgraded generation of phospholipids for drug delivery’ by Dali Wang  DMA and DOA were displayed incorrectly in the graphical abstract and Graphical abstract:The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.


The inhibitory effect of dAbs on TNF-α biological effect was determined in MTT assay in which I44 and I49 prevented TNF-α cell cytotoxicity with IC50 values of 6.61 and 3.64 µM, respectively. The identified anti-TNF-α dAbs could bind to and inhibit TNF-α activity. The dAbs activities can be attributed to their ability to establish hydrogen bonds as well as hydrophobic contacts with TNF-α. The results of the current study can pave the way for further structural studies in order to introduce new more potent anti-TNF-α antibodies.Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine which plays crucial roles in pathogenesis of inflammatory diseases. The current study aimed to investigate the binding abilities of I44 and I49 domain antibodies to TNF-α. The dAbs were expressed in bacterial expression system and purified by affinity chromatography using Ni-sepharose column. The expression and purity of the proteins were evaluated using western blotting and SDS-PAGE techniques, respectively. ELISA experiment showed that I44 and I49 dAbs bind to TNF-α with the binding constants (K To gain insight into the mode of interactions of the identified anti-TNF-α dAbs with TNF-α, molecular docking study was performed. Cytokines, as a result of inflammation, lie at the heart of chronic inflammatory and autoimmune diseases. Tumor necrosis factor alpha (TNF-α) is one of the crucial cytokines, as it directly initiates the inflammatory processes and also activates the other inflammatory cytokines . Althougic cells . TNF-α iic cells , 4. At tic cells . Rheumatic cells . Beside ic cells , 11. Theic cells -16. Althic cells , 17-19. ic cells . Antibodic cells , 22. In ic cells . The purReagents Tryptone, yeast extract, isopropyl-D-thio galactopyranoside (IPTG), Triton X-100, potassium acetate, phenylmethylsulfonyl fluoride (PMSF), and N,N,N′,N′-tetra methyl ethylene diamine (TEMED), were purchased from AppliChem . 3,3′,5,5′-Tetramethylbenzidine (TMB),Acrylamide, N,N′-methylene-bis-acrylamide, and thiazolyl blue tetrazolium bromide (MTT) were obtained from Sigma Aldrich, USA. Mouse anti-His primary antibody was prepared from GE Healthcare (Sweden). Goat anti-mouse IgG-HRP secondary antibody was purchased from Santa Cruz Biotechnology (USA). BM Chemiluminescence Western Blotting kit was purchased from Roche Diagnostics GmbH . Adalimumab was a kind gift from CinnaGen Company, Iran. All chemicals and reagents were of molecular biology grade. Ultra pure water, produced by Milli-Q system , was used for preparation of all solutions. Expression and purification of anti –TNF-α domain antibodiesTomlinson I and J scfv libraries were used for identification of specific anti-TNF antibodies as described in our previous study in which phage harboring I44 and I49 dAbs demonstrated high affinity towards TNF-α . E. coli origami(DE3) was infected by phage particles harboring I44 and I49 coding sequences. To do this, from each serially diluted phage, 10 µL was added to 100 µL E. coli origami(DE3) at OD 0.4 followed by incubation at 37 °C for 30 min without shaking. Then the mixtures were poured onto LB plates supplemented with 100 µg/mL ampicillin and 1% glucose. In the next day, a single colony of E. coli origami (DE3), infected with phage, was inoculated into 10 mL LB-ampicillin medium and cultured overnight at 37 °C. The overnight culture was diluted 1:50 in 500 mL LB medium and incubated at 37 °C while shaking. At OD of 0.9, IPTG with final concentration of 1 mM, was added and the temperature was set to 30 °C for overnight incubation. Then the culture was centrifuged at 3,000×g for 15 min and the harvested bacterial pellet was resuspended in lysis buffer . The suspension was freeze-thawed three times using liquid nitrogen followed by sonication five times on ice at 60% pulse for 30 sec with 30 sec intervals. Bacterial debris was removed by centrifugation at 8,0002PO4 pH 7.4, and NaCl 500 mM. Finally, the purified proteins were dialyzed against Tris 50 mM pH 8, NaCl 150 mM buffer, and analyzed on SDS-PAGE to monitor the protein production and purification. Western blotting technique was used to verify the expression of antibodies in which mouse anti-His primary antibody (1:3000 diluted in BSA) and goat anti-mouse IgG-HRP secondary antibody (1:8000 diluted in BSA) were used for protein detection. The protein bands were visualized by applying enhanced chemiluminescence (ECL) reagent.In order to express I44 and I49 proteins, ELISA experiment2SO4. The absorbance was measured at 450 nm and 650 nm using ELISA plate reader. TNF-α uncoated wells and TNF-α coated wells without addition of dAb were used as the controls. All data points were the average of three individual experiments. The results were fit into one-site specific binding curve using Prism program  in order to calculate the Kd values.The functionality of the produced dAbs was tested by ELISA experiment. To this end, TNF-α (100 µg/mL) prepared in house as described previously in , was coaMTT assay2. Different concentrations of the affinity purified dAbs (ranging from 0.023 to 1.83 µM for I44 and 0.047 to 3.82 µM for I49) were added to 0.0057 nM of TNF-α and the mixtures were incubated for 24 h in the presence of Actionomycin (1 µg/mL) at 37 °C. The mixtures were then added to the seeded cells and incubated for additional 24 h. Adalimumab, a marketed anti-TNF-α antibody at different concentration between 6.76E-5 and 6.76 nM, were used as the positive control. After incubation, 20 µL of MTT (5 mg/mL) was added to each well and the plate was kept in the incubator for 4 h. After discarding the supernatant, 100 µL of solublizing buffer containing Sorensen buffer (12.5%) and DMSO (87.5%) was added to each well and incubated for 40 min at room temperature with agitation. Finally, the absorbance was measured at 570 nm. For each concentration the experiment was carried out in triplicate wells. The achieved results represented as the percentages of TNF-α neutralizing effects in comparison to the TNF-α treated (maximum toxicity) and untreated cells (maximum viability) were fit into dose-response inhibition curve using Prism program .Murine L929 fibroblast cells were cultured in RPMI supplemented with 10% FBS. The cells were harvested in log phase growth using trypsin-EDTA. L929 cell suspension including approximately 10,000 cells were seeded in each well of 96-well plate and incubated for 24 h at 37 °C under a humidified atmosphere and 5% COMolecular modeling and docking studyThe crystal structure of TNF-α trimer was retrieved from protein data bank (PDB ID: 1TNF). In order to construct the 3D-structure of I44 and I49 dAbs from the corresponding amino acid sequences , the homExpression and purification of anti –TNF-α domain antibodiesE. coli origami(DE3) expression system induced by 1mM IPTG under temperature of 30 °C for overnight. The produced I44 and I49 antibodies were purified on affinity column resulting in 0.28 and 0.6 mg purified proteins, respectively  in different concentrations ranging from 0.35 to 5.83 µM were added to 96-well plate coated with TNF-α. Then, anti-6×His and HRP-conjugated goat anti-mouse antibodies were sequentially used to evaluate the TNF-α binding ability of dAbs. Analyzing of the results showed that I44 and I49 dAbs bind to TNF-α with Kd values of 5.18 ± 1.41 and 2.42 ± 0.55 µM, respectively  were identified (E.coli origami (DE3) bacteria. Presence of PelB signal peptide in phage genome enables periplasmic expression of the target proteins . This feature provides an advantage for the current expression system where periplasmic compartment supplies a reduced environment suitable for the formation of disulfide bonds in 3D structures of I44 and I49 proteins . Such big difference in IC50 values can be attributed to the lower binding valence of I44 and I49 single dAbs (~17 kDa) compared to adalimumab (148 kDa). To identify the most important residues involved in antibodies-TNF-α interactions, molecular docking was carried out in which the modeled structures of dAbs were docked onto TNF-α trimer. The docking scores obtained from ZDock program were 952.23 and 981.76 for I44-TNF-α and I49-TNF-α complexes, respectively, which is in close agreement with the data achieved from ELISA experiment where I49 showed higher affinity towards TNF-α compared to I44. In the next step, the possible interactions between dAbs and TNF-α were predicted using PIC web server  and I49 Tyr53 (CDR2) with Val91 and Gly148 of TNF-α, respectively (Progress of inflammatory conditions greatly depends on TNF-α aberrant production. Therefore, prevention of TNF-α activity would result in promising outcomes in patients suffering from inflammatory diseases. In this regard much attention has been devoted to develop anti-TNF-α agents which could directly bind to TNF-α suppressing its adverse effects. These inhibitory molecules vary from small molecules such as peptides to macromolecules such as full length antibodies -42. Up tentified . Compareentified . On the entified . Based oproteins , 55. Thiproteins . The varproteins . In ELISas scFvs , 58. Theb server . Accordiof TNF-α . In addiof TNF-α . These rucidated . Apart fectively . Such inectively . Compariectively . Compariectively , 61. In the current study, the previously identified anti-TNF-α dAbs, I44 and I49, were recombinantly produced and purified. The binding properties of the prepared antibodies were determined using experimental and molecular modeling techniques. Based on experimental assays, these dAbs are capable of binding to and inhibiting TNF-α cytotoxic effects. Molecular docking studies identified the key residues important in the interactions between the studied domain antibodies and TNF-α. The information obtained in the current study can be useful for further structural studies and designing potent anti-TNF-α small format antibodies.


Osteocytes cultured with TNF-α show up-regulation of MAPKs phosphorylation measured by western blot, and adding MAPKs inhibitors to osteocytes cultured with TNF-α significantly decreases RANKL mRNA expression compared to osteocytes cultured with TNF-α alone. We also found that TNF-α activates the NF-κB pathway in osteocytes measured as a function of p65 subunit nuclear translocation. TNF-α directly affects osteocyte RANKL expression and increases osteoclastogenesis; our results demonstrate that osteocytes guard an important role in inflammatory bone resorption mediated by TNF-α.Osteoimmunology peeks into the interaction of bone and the immune system, which has largely proved to be a multiplex reaction. Osteocytes have been shown to regulate bone resorption through the expression of RANKL in physiologic and pathologic conditions. TNF-α, a product of the immune system, is an important cytokine regulating bone resorption in inflammatory conditions either directly or by increasing RANKL and M-CSF expressions by osteoblasts and stromal cells. The effect of TNF-α on a wide range of cell types has been documented; however, the direct effect of TNF-α on osteocytes has not been established yet. In this study, primary osteocytes were isolated by cell sorting from neonatal calvaria of Dmp1-Topaz mice, which express the green fluorescent protein under the influence of dentin matrix protein 1 promoter. The results show that osteocytes have a significantly higher RANKL mRNA expression when cultured with TNF-α. A co-culture system of osteocytes and TNF receptors I and II deficient osteoclast precursors treated with TNF-α show a significant increase in TRAP-positive cells while cultures without TNF-α failed to show TRAP-positive cells. Additionally,  The bone is a dynamic tissue, it is continuously broken down and built through the process of bone remodeling in which bone cell populations serve to achieve a balance between episodes of resorption and deposition . OsteoclMolecular signals act together with cellular components to regulate bone resorption, macrophage colony-stimulating factor (M-CSF) is the first in line to induce osteoclastogenesis by binding to its c-fms receptor to promote differentiation and maturation of osteoclast precursors . ReceptoOsteocytes are terminally differentiated osteoblasts which became embedded in their secreted matrix , 16 and Pasteurella multocida toxin resulted in a decreased number of RANK positive cells, a marker of osteoclasts  mice and C57BL/6-Tg(Dmp1-Topaz)1Ikal/J mice were purchased from The Jackson Laboratory . All animal procedures were performed in accordance with Tohoku University regulations. Recombinant murine TNF-α was prepared in our laboratory as described previously (Eight-week-old C57BL6/J wild-type (WT) were purchased from CLEA Japan Inc. . B6.129S-Tnfrsf1aeviously . Recombieviously .2HPO4, 1 mM CaCl2, 0.1% (w/v) BSA, 0.5% (w/v) glucose and 25 mM HEPES. Calvariae were incubated in collagenase for 20 min or EDTA for 15 min at 37°C on a shaker as follows: fraction 1 (collagenase), fraction 2 (EDTA), fraction 3 (collagenase), fraction 4 (collagenase), and fraction 5 (EDTA). Fractions 2 through 5 were collected and cultured in α-MEM  containing 10% fetal bovine serum (FBS) , 100 IU/ml penicillin G, and 100 μg/ml streptomycin overnight. Fraction 2 was used in subsequent experiments as an osteoblast high fraction. Adherent cells were harvested using trypsin-EDTA  and strained through a 40 μm nylon cell strainer  in preparation for fluorescence-activated cell sorting in FACSAriaTM II . GFP positive and negative cells were visualized using a fluorescence microscope  and the purity of osteocytes was determined through real-time RT-PCR.To isolate osteocytes of high purity, we followed the protocol published by Halleux et al. , 27 with® IV First-Strand Synthesis System  according to the manufacturer's instructions. mRNA expression values were measured using the Thermal Cycler Dice Real-Time System . Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a reference gene. The following primers were used: RANKL (5′-CCCATCGGGTTCCCATAAAGTC-3′), (5′-GCCTGAAGCAAATGTTGGCGTA-3′), OPG (5′-ATCAGAGCCTCATCACCTT-3′), (5′-CTTAGGTCCAACTACAGAGGAAC- 3′), GAPDH (5'-GGTGGAGCCAAAAGGGTCA-3′), (5′-GGGGGCTAAGCAGTTGGT-3′), M-CSF(5′-TGATTGGGAATGGACACCTG-3′), (5′-AAAGGCAATCTGGCATGAAGT-3′). The purity of osteocytes obtained through cell sorting was checked against the following primers: Dmp1(5′-ACCACACGGACAGCAGTGAATC-3′), (5′-CCTCATCGCCAAAGGTATCATCTC-3′), SOST (5′-AGCCTTCAGGAATGATGCCAC-3′), (5′-CTTTGGCGTCATAGGGATGGT-3′), while osteoblast rich fraction content (fraction 2) was tested against Kera (5′-TCCCCCATCAACTTATTTTAGC-3′), (5′-GGTTGCCATTACAGGACCTT-3′).Osteocytes were cultured in α-MEM with or without TNF-α 100 ng/ml for 3 days. To test the effect of MAPKs inhibition, Osteocytes were pre-incubated with 10 μM of SB 203580  a p38 MAPK inhibitor, 10 μM of U0126  a MEK1/2 inhibitor targeting ERK1/2 MAPK, and 10 μM of JNK Inhibitor II  an inhibitor of JNK I, II, and III. Total RNA was obtained from lysed cells by using RNeasy Minikit  according to manufacturer's instructions. Total RNA was used to synthesize cDNA using the SuperScript4/well were cultured overnight in α-MEM and harvested WT or TNFR I, II deficient osteoclast precursors 5 × 104/well were added on top of osteocytes (2:1), and the co-culture was maintained either without M-CSF, with M-CSF alone (100 ng/ml), TNF-α alone (100 ng/ml), M-CSF+TNF-α (100 ng/ml), or M-CSF+RANKL 100 ng/ml in a 96 well plate. To test the effect of OPG on osteoclastogenesis, osteocytes and TNFR I, II deficient osteoclast precursors were co-cultured with M-CSF+TNF-α (100 ng/ml) or M-CSF+TNF-α+OPG (100 ng/ml), OPG was purchased from R&D Systems . To test the significance of cell-to-cell contact, an osteocyte-conditioned medium was prepared as follows: osteocytes were cultured with M-CSF+TNF-α (100 ng/ml) for 1 day, the medium was then aspirated and added to TNFR I, II deficient osteoclast precursor, as a positive control TNFR I, II deficient osteoclast precursor were cultured with M-CSF+RANKL (100 ng/ml). The co-cultures were maintained for 4 days after which they were terminated by fixation with 4% paraformaldehyde and stained with tartrate-resistant acid phosphatase (TRAP) staining consisting of acetate buffer (pH 5.0), naphthol AS-MX phosphate, fast red violet LB salt, and 50 mM sodium tartrate. Cells were considered osteoclasts if they were TRAP-positive and had 2 or more nuclei.The bone marrow of long bones of WT and TNFR I, II deficient mice was flushed and cultured in α-MEM containing 10% FBS, 100 IU/ml penicillin G, 100 μg/ml streptomycin, and M-CSF 100 ng/ml for 4 days. Attached cells were harvested by trypsinization and used as osteoclast precursors. Osteocytes 2.5 × 10® Simple StainTM Mouse MAX PO (R)  for 1 h at room temperature then stained with hematoxylin as a counterstain. RANKL-positive osteocytes were counted as a percentage from the total number of osteocytes per 400 × 400 μm2 section area. For osteoclast formation examination, calvarial sections from WT mice were deparaffinized, rehydrated, and stained with TRAP staining then counter-stained with hematoxylin. TRAP-positive cells with two or more nuclei were counted and corrected to total bone surface area.WT mice were subjected to subcutaneous supra-calvarial injection of either TNF-α 3.0 μg/100 μl or PBS for 5 consecutive days, on the 6th day the mice were sacrificed and the calvaria was excised. The calvaria was fixed in 4% paraformaldehyde overnight at 4°C and demineralized in 14% EDTA for 3 days at room temperature. The calvaria was trimmed from soft tissue and cut into 3 pieces perpendicular to the sagittal suture, and dehydrated then embedded in paraffin. Paraffin-embedded calvaria specimens were cut into 5 μm-thick sections using a microtome . Histological sections were deparaffinized, rehydrated, and blocked with 3% skimmed milk for 30 min at 37°C then stained with anti-RANKL antibody  diluted to 1:50 in can get immunostain solution B  overnight at 4°C. Sections were washed and incubated with HistofineOsteocytes were cultured in α-MEM containing 10% FBS, 100 IU/ml penicillin G, 100 μg/ml streptomycin overnight. Osteocytes were then cultured in a 24-well plate in α-MEM containing no serum (serum starvation) for 3 h. TNF-α 100 ng/ml was then added to the wells for specific periods  minutes. Control wells (0 min) had no TNF-α. Cells were lysed using radioimmunoprecipitation (RIPA) assay buffer  containing 1% protease and phosphatase inhibitor  on ice for 15 min; insoluble material was separated by centrifugation. Total protein was quantified using Pierce BCA protein assay kit . Protein was treated with β-mercaptoethanol  and laemmli sample buffer  1:1 and denatured at 95°C for 5 min as preparation for SDS-PAGE. Equal amounts of protein were loaded into gels 4–15% Mini-PROTEAN TGX Precast Gels  and transferred to a PVDF Trans-Blot Turbo Transfer System  then incubated in Block-Ace  at 4°C overnight. Membranes were incubated with the following antibodies: rabbit monoclonal Phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK rabbit Ab, rabbit monoclonal Phospho-p44/42 (ERK1/2) MAPK (Thr202/Tyr204), p44/42 (ERK1/2) MAPK rabbit Ab, polyclonal Phopho-SAP/JNK (Thr183/Tyr185), SAP/JNK MAPK rabbit Ab, polyclonal Phospho-AKT (Ser473), AKT rabbit Ab , mouse monoclonal anti-β-actin antibody  at a dilution 1:1,000 overnight at 4°C. The membranes were washed in tris buffered saline with Triton X-100 (TBS-T) and incubated with horseradish peroxidase-conjugated anti-rabbit antibody  or anti-mouse antibody  at a dilution 1:5,000 for 1 h at room temperature. The signal was detected using an enhanced chemiluminescence detection system . Band density was measured using Image J (NIH).TM II .Bone marrow cells (BMC) from WT mice and fractions 2–5 from Dmp1-topaz mouse calvaria were suspended in α-MEM 100 IU/ml penicillin G, 100 μg/ml streptomycin. Cells were washed with 1% BSA in PBS, fixed in 4% paraformaldehyde for 3 min, and washed with 1% BSA in PBS 3 times. Cells were incubated with PE-conjugated anti-TNFR I antibody  at 1 μg/ml and PE-conjugated anti-TNFR II  at 1 μg/ml for 30 min on ice in the dark and washed 3 times with 1% BSA in PBS. Stained cells were analyzed with FACSAriaOsteoblasts from fraction 2 and osteocytes were cultured overnight in α-MEM 100 IU/ml penicillin G, 100 μg/ml streptomycin. Cells were washed with PBS, fixed with 4% formaldehyde for 15 min, washed 3 times with PBS and blocked with 3% BSA in PBS for 1 h. Cells were incubated with PE-conjugated anti-TNFR I antibody , and its isotype control  at 1 μg/ml and PE-conjugated anti-TNFR II , and its isotype control  at 1 μg/ml on ice in the dark for 1 h. Cells were washed 3 times with PBS before imaging. For testing the NF-κB pathway activation, osteocytes were cultured on collagen-coated  8-chamber slide  with or without TNF-α 100 ng/ml for 1 h. Cells were washed with PBS, fixed in 4% formaldehyde for 15 min and washed, then permeabilized with 0.1% Triton X-100 in PBS for 10 min, washed and blocked with 3% BSA in PBS for 1 h at room temperature (R/T). Cells were then incubated with anti-p65 antibody at (1:100) in 3% BSA  at 4°C overnight. Cells were washed with PBS and incubated with Alexa Fluor 555 (goat anti-rabbit IgG) (1:400) in 3% BSA  for 1 h in the dark at R/T, then washed and incubated with DAPI for 5 min. Fluorescent cell imaging was done using a fluorescence microscope .t-test for two data sets and scheffe's test for multiple data sets. Data were presented as a mean ± standard deviation (SD). Results are representative of three or more independent experiments throughout the paper.The data were analyzed using student's In order to extract osteocytes from their calcified bony matrix, we processed the calvaria using collagenase and EDTA, and then we run the digests through FACS to separate GFP-positive from GFP-negative populations . The isoFlow cytometry analysis shows that osteocytes (GFP+) stained for TNFR I and II express both receptors . UnstainWe examined the change in RANKL, OPG, and M-CSF expression levels in osteocytes upon incubation of osteocytes with TNF-α for 3 days. PCR analysis revealed that osteocytes cultured with TNF-α expressed a significantly higher RANKL mRNA  but no dTo test whether osteocytes cultured with TNFR I, II-deficient osteoclast precursors in the presence of TNF-α successfully supports the generation of multinuclear TRAP-positive osteoclasts; we cultured osteocytes and TNFR I, II-deficient osteoclast precursor with TNF-α or without TNF-α in the presence of M-CSF. While osteocytes were able to induce osteoclast formation in TNF-α+M-CSF treated wells, osteoclastogenesis failed without the addition of M-CSF even with the addition of TNF-α . Co-cultTRAP staining of calvaria revealed a higher number of osteoclasts in TNF-α injected calvaria compared to PBS injected calvaria . ImmunohTNF-α was added to cultures of osteocytes for specific time intervals  minutes, while 0 indicates that no TNF-α was added. TNF-α transiently increased the phosphorylation of ERK1/2, p38, and JNK MAPKs in relation to the corresponding total protein and actin, peaking at 5 or 15 minutes . There wTo evaluate NF-κB pathway activation, we relied on immunofluorescence imaging of osteocytes cultured with TNF-α . We assein vitro and in vivo.Osteocytes have gained popularity ever since their isolation from bone became possible , from thFirst, we obtained pure cultures of primary osteocytes by cell sorting. To test the purity of the isolated population, we selected Dmp1, a gene predominantly expressed by osteocytes and was reported to exhibit the highest expression level in osteocytes compared to osteoblasts, and Kera, a gene which encodes keratocan and has the highest change in negative expression as osteoblasts mature into their osteocytic phenotype, Dmp1 and Kera were reported to have the highest difference in their expression levels between osteocytes and osteoblasts amongst genes associated with extracellular matrix and secreted proteins .We also 2 (PGE2) and 1,25 dihydroxy vitamin D3 , their conclusion was that despite the high expression of RANKL, downregulation of OPG expression is required for efficient osteoclastogenesis (Two cell surface receptors, p55 and p75 TNFRs mediate the action of TNF-α . We testogenesis . Our resogenesis .in vitro results also show that osteocytes cultured with TNF-α for 3 days exhibit no difference in M-CSF mRNA expression. Our interest in M-CSF expression in osteocytes stems from the findings of previous studies which noted that TNF-α increases the pool of osteoclast precursors available due to an increase in M-CSF production in stromal cells (Osteocytes co-cultured with TNFR I, II deficient osteoclast precursor failed to form osteoclasts without M-CSF, even with the addition of TNF-α, which indicates that osteocytes are not a significant source of M-CSF. Our al cells , and thaal cells  and in tal cells , both ofin vivo and in vitro by regulating MAPKs phosphorylation and possibly NF-κB activation. TNF-α is a potent activator of ERK, JNK and p38 MAPKs, and their interaction with TNF-α has functions upstream and downstream of TNF-α signaling (TNF-α was shown to increase the expression of RANKL in osteoblasts, stromal cells  and lympignaling . In thisignaling , respectNF-κB is involved in the transcriptional activation of inflammatory-related genes induced by TNF-α, and its activation is crucial for the protection of cells from apoptotic cell death induced by the TNFR I cytoplasmic death domain activation. In an unstimulated state, IκB proteins interact with NF-κB units to mask their nuclear translocation, in this report TNF-α significantly increased the nuclear presence of the NF-κB p65 unit in osteocytes nuclei after 1 h of treatment. AKT is also a pathway when activated works as an antiapoptotic signal, TNF-α stimulates the AKT pathway in a cell-type-specific manner , we founThe results of this study can be summarized by asserting the role of osteocytes in their potential contribution to bone destruction in inflammatory bone diseases by increasing RANKL expression. Osteocytes could be a target therapeutic factor in future studies.All datasets generated for this study are included in the article/The animal study was reviewed and approved by Animal Care and Use Committee of Tohoku University.AM and HK contributed to conception, design, data acquisition, data analysis, data interpretation, and drafting of the manuscript. HK contributed to critical revision of the manuscript. FO, AK, SO, W-RS, JQ, TN, and YN collected the samples and performed data analyses. HK and IM supervised the project. All authors provided final approval and agreed to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


G-protein-coupled receptor 40 (GPR40) has an anti-apoptotic effect in pancreatic β-cells. However, its role in renal tubular cell apoptosis remains unclear. To explore the role of GPR40 in renal tubular apoptosis, a two-week unilateral ureteral obstruction (UUO) mouse model was used. The protein expression of GPR40 was decreased, while the Bax/Bcl-2 protein expression ratio, the expression of tumor necrosis factor (TNF)-α mRNA, and angiotensin II type 1 receptor (AT1R) protein were increased in mice with UUO. In vitro, pretreatment of rat proximal tubular (NRK52E) cells with GW9508, a GPR40 agonist, attenuated the decreased cell viability, increased the Bax/Bcl-2 protein expression ratio, increased protein expression of cleaved caspase-3 and activated the nuclear translocation of nuclear factor-κB (NF-κB) p65 subunit induced by TNF-α treatment. TNF-α treatment significantly increased the expression of AT1R protein and the generation of reactive oxygen species (ROS), whereas GW9508 treatment markedly reversed these effects. Pretreatment with GW1100, a GPR40 antagonist, or silencing of GPR40 in NRK52E cells promoted the increased expression of the cleaved caspase-3 protein by TNF-α treatment. Our results demonstrate that decreased expression of GPR40 is associated with apoptosis via TNF-α and AT1R in the ureteral obstructed kidney. The activation of GPR40 attenuates TNF-α-induced apoptosis by inhibiting AT1R expression and ROS generation through regulation of the NF-κB signaling pathway. G-protein-coupled receptor 40 (GPR40), also known as free fatty acid receptor 1, is a cell surface receptor highly expressed in pancreatic β-cells, intestine and enteroendocrine cells of the gastrointestinal tract, taste cells, immune cells, splenocytes, and brain cells ,3,4. We GPR40 couples with a G protein α-subunit of the Gq family , and itsTubular atrophy is the common characteristic feature of chronic kidney disease (CKD) and is superior to glomerular pathology as a predictor of CKD progression . TubularIn this study, we determined whether the expression of GPR40 was changed in a ureteral obstructed kidney model and was associated with apoptosis. Furthermore, we investigated the effects of GPR40 activation on the pathogenesis of apoptosis induced by TNF-α treatment in rat proximal tubular (NRK52E) kidney cells.Following unilateral ureteral obstruction (UUO) for 2 weeks, expression of the GPR40 protein was significantly decreased, while the Bax/Bcl-2 protein ratio was increased in the obstructed kidney compared with that in the control .Immunoreactive GPR40 was expressed in renal tubules of the control kidney. However, decreased immunostaining of GPR40 with tubular atrophy was observed in the ureteral obstructed kidney A.Immunofluorescence staining revealed decreased immunoreactivity of GPR40 in aquaporin-1 (AQP1)- and AQP2-positive renal tubules of the ureteral obstructed kidney B. In addNext, we performed in vitro studies to explore the effect of GPR40 on apoptosis and the downstream signaling pathways activated in response to TNF-α. The WST-1 assay revealed that treatment of NRK52E cells with TNF-α (20 ng/mL) decreased their viability compared with vehicle-treated cells. Pretreatment with GW9508, a small-molecule GPR40 agonist, attenuated the decreased cell viability caused by TNF-α A. TreatmAlterations in AT1R protein expression, TNF-α mRNA levels and ROS generation were examined to determine the mechanisms of TNF-α-induced tubular injury. All of these factors were increased by treating NRK52E cells with TNF-α. Notably, we observed that these TNF-α-stimulated changes were attenuated by pretreatment with GW9508 A–C.Lastly, pretreatment with GW1100, a GPR40 antagonist, or silencing GPR40 in NRK52E cells with siRNA, markedly augmented the increased levels of cleaved caspase-3 caused by TNF-α treatment A,B. ThesUrinary tract obstruction has been recognized as an experimental model of CKD because the same cellular and molecular events that characterize the progression of CKD also occur in the ureteral obstructed kidney of rats and mice with UUO ,20. TubuGPR40 was identified as an orphan 7-transmembrane G-protein-coupled receptor . It funcThe GPR40 protein and mRNA are expressed in the renal tubular epithelial cell line, LLCPKcl4, and mouse kidney, as well as the pancreas and the brain . ImmunobIn the present study, we demonstrated that the expression of the GPR40 protein was decreased in the ureteral obstructed mouse kidney compared with the control kidney, and was related to the increased Bax/Bcl-2 protein ratio and tubular atrophy. The GPR40 protein was expressed in both AQP1- and AQP2-positive renal tubules. These results suggest that a decreased renal tubular expression of GPR40 may be related to the pathogenesis of apoptotic tubular injury in the ureteral obstructed kidney.Ureteral obstruction induces the production of renal TNF-α and AT1R, renal tubular cell apoptosis, caspase activity, and NF-κB activity, and increases Bax and decreases Bcl-2 expression, all of which are ameliorated by neutralizing TNF-α ,24,25. TConsistent with these findings, in our study, the expression of TNF-α mRNA and the AT1R protein was increased in the ureteral obstructed kidney. Furthermore, our in vitro results show that the expression of the AT1R protein and TNF-α mRNA, and the generation of ROS, were increased in TNF-α-treated NRK52E cells. Nuclear translocation of the NF-κB p65 subunit also increased following TNF-α treatment. Therefore, TNF-α treatment resulted in increased cell death and the activation of the apoptotic signaling pathway in rat proximal tubular cells .Overall, our results demonstrate that GPR40 activation rescued cell death and activated the pro-apoptotic signaling pathway in TNF-α-induced injury of NRK52E cells. In addition, the activation of GPR40 inhibited TNF-α-mediated NF-κB activation. The inhibition of GPR40 by a GPR40 antagonist, GW1100, or GPR40 siRNA transfection, promoted the expression of pro-apoptotic proteins by TNF-α treatment. These observations suggest that GPR40 has an anti-apoptotic effect in renal tubular epithelial cells. Our findings raise the possibility that GPR40 might be a novel target for the prevention and treatment of renal cell apoptosis.The experimental protocol was approved by the Institutional Animal Care and Use Committee of Chonnam National University Medical School . Male 8-week-old C57BL/6J mice were used . The mice were randomly assigned to the control group and the UUO group, with eight mice each group. UUO was induced by ligation of the left proximal ureter as previously described .The NRK52E cells  were treated with TNF-α  for 24 h in the presence or absence of the GPR40 agonist, GW9508 , for 1 h prior to the addition of TNF-α, and then harvested for further analysis. The control cells were treated with the vehicle (dimethyl sulfoxide). The NRK52E cells were also treated with TNF-α with or without a 1 h pretreatment with the GPR40 antagonist, GW1100 , or GPR40 small interfering (si)RNA , Item No. L-080051-02-0010, Dharmacon, Lafayette, CO, USA).Cell viability was determined using the EZ-CyTox  cell viability assay kit , as previously described . Absorba® nuclear and cytoplasmic extraction reagent  according to the manufacturer’s protocol, as previously described [g for 2 min. After removing the supernatant, 100 µL of ice-cold cytoplasmic extraction reagent I was added to the cell pellets and then incubated on ice for 10 min. The ice-cold cytoplasmic extraction reagent II was then added to the tube and centrifuged at 16,000× g for 5 min. The pellet was suspended in 50 µL of ice-cold nuclear extraction reagent, followed by centrifugation at 16,000× g for 10 min. Finally, the supernatant containing the nuclear extract was transferred to a new tube and the protein concentration was measured.To prepare nuclear extracts, the cells were lysed using the NE-PERescribed . Briefly2DCF-DA) fluoroprobe . The NRK52E cells were incubated with 5 µM H2DCF-DA for 30 min at 37 °C. Then, the cells were washed, collected by centrifugation, and resuspended in PBS. The fluorescence intensity was measured using a FACSCalibur™ flow cytometer .Intracellular ROS generation was measured with a 2′,7′-dichlorodihydrofluorescein diacetate (HAnti-Bax (2772), anti-Bcl-2 (2870), anti-caspase-3 (9662), anti-cleaved caspase-3 (9661), anti-NF-κB p65 subunit (3034), anti-histone H3 (9715), and anti-IκB-α (9242) antibodies were purchased from Cell Signaling Technology . The anti-GPR40 and anti-AT1R (SC-1173) antibodies were obtained from Santa Cruz Biotechnology . An anti-β-actin (A3854) antibody  was used as the control.Primer sequences for RT-PCR were as follows: for TNF-α, 5′-GTC GTA GCA AAC CAC CAA GC-3′ (forward) and 5′-CTC CTG GTA TGA AAT GGC AAA-3′ (reverse); for GAPDH, 5′-ATC AAA TGG GGT GAT GCT GGT GCT G-3′ (forward) and 5′-CAG GTT TCT CCA GGC GGC ATG TCA G-3′ (reverse).The kidneys, fixed in 4% paraformaldehyde, were dehydrated through a graded series of ethanol, embedded in paraffin, sectioned (5 µm), and mounted on glass slides. After deparaffinization and rehydration, antigen retrieval was performed by using Antigen Unmasking Solution . Sections were blocked with 2.5% bovine serum albumin in PBS, incubated with the anti-GPR40 antibody overnight at 4 °C, and then with the appropriate secondary antibody. For immunofluorescence labeling with specific tubular maArkers, the sections were incubated with an Alexa Fluor 568-labeled goat anti-rabbit IgG  secondary antibody after incubation with the anti-GPR40 antibody. Anti-AQP1 and anti-AQP2 antibodies  were used as markers for the proximal tubule and for the collecting duct, respectively. The sections were incubated with FITC-conjugated secondary antibodies and the nuclei were counterstained with 4′,6-diamidino-2-phenylindole. Images were captured using an LSM 510 confocal microscope .p values were <0.05, using the GraphPad Prism 6 .The results are expressed as means ± standard error of the mean. The statistical significance of the differences was determined by the unpaired t-test or one-way analysis of variance followed by the post-hoc Tukey’s HSD (honestly significant difference) test. The differences were considered statistically significant when the The results of our study demonstrate that the decreased expression of GPR40 in the ureteral obstructed kidney is associated with apoptosis via the activation of TNF-α and AT1R. In addition, the activation of GPR40 attenuates TNF-α-induced apoptosis by inhibiting AT1R expression and ROS generation through the regulation of NF-κB signaling pathways.


ATG16L1 with susceptibility to inflammatory bowel disease (IBD) have prompted mucosal immunologists to investigate the functional roles of macroautophagy/autophagy in different cell types in the gut. Here we present a recent study that addressed 2 key questions: in which cell type is autophagy deficiency most detrimental during chronic colitis and what is the functional role of autophagy in those cells? We report that autophagy in intestinal epithelial cells (IECs) acts to limit intestinal inflammation by protecting them from TNF-induced apoptosis and we discuss the potential implications for IBD treatment.Genome-wide association studies (GWAS) linking polymorphisms in  ATG16L1 and IRGM, have been linked to IBD by GWAS. Autophagy is a versatile cellular degradation pathway implicated in many stress response mechanisms fundamentally important in nearly every cell type. Therefore, the genetic links identified by GWAS do not give any mechanistic information about the functional consequences, or about the cell types in which the autophagy pathway is important during chronic intestinal inflammation.Inflammatory bowel disease is a complex inflammatory disorder of the gastrointestinal tract thought to arise from a dysregulated immune response towards the microbiota. Although many pathogenic networks underlying disease development have been identified, the exact etiology is unknown, reflecting the complexity and diversity of the disease manifestations. Polymorphisms in several autophagy-related genes, such as Atg16l1 was selectively ablated in distinct cellular compartments. To assess the impact on chronic intestinal inflammation, we used an IBD model triggered by infection with the opportunistic bacterial pathogen Helicobacter hepaticus and simultaneous inhibition of the regulatory arm of the immune response with anti-IL10R blocking antibody. Although previous studies in other systems suggest that autophagy can limit release of pro-inflammatory cytokines by myeloid cells (macrophages and dendritic cells), we found that selective ablation of Atg16l1 in the myeloid compartments has little impact on this bacterially triggered chronic intestinal inflammation. In contrast, mice with selective ablation of autophagy in IECs (Atg16l1VC mice) exhibit severely exacerbated intestinal pathology, characterized by increased accumulation of CD4+ T cells in the lamina propria and elevated levels of pro-inflammatory cytokines.Recently, we addressed this issue by utilizing a Cre-lox genetic approach to generate transgenic mouse lines in which the IBD-associated autophagy gene Atg16l1VC mice is also accompanied by a higher number of apoptotic cells in the epithelium. Increased epithelial cell death could result in impaired barrier function leading to exacerbated pathology; however, it could also be just a consequence of the elevated inflammatory cytokines present in Atg16l1VC mice. This led us to investigate whether autophagy intrinsically regulates cytokine-induced apoptosis in IECs. Using complementary approaches, we found that autophagy directly controls apoptosis in epithelial cells. First, utilizing the recently developed ‘organoid’ system to culture primary IECs ex vivo, we found that Atg16l1-deficient IECs show increased induction of apoptosis following exposure to pro-inflammatory cytokines (TNF + IFNG/IFNγ) compared to wild-type IECs. Second, in vivo injection of TNF induces higher numbers of apoptotic IECs in the small intestine of Atg16l1VC mice compared to wild-type mice. Of note, increased apoptosis in autophagy-deficient IECs is only observed following inflammatory cytokine stimulation or in the context of chronic inflammation; at steady-state, IECs cope well with the lack of autophagy in regard to proliferation and differentiation, and exhibit normal inflammatory responses.The increased pathology in Atg16l1VC mice. Indeed, TNF blockade significantly attenuates disease severity and reduces IEC apoptosis in Atg16l1VC mice. These findings confirm that the exacerbated pathology in Atg16l1VC mice is largely driven by TNF-induced IEC apoptosis (It has been previously recognized that TNF induces IEC apoptosis in the context of IBD and in murine disease models. Our results show that autophagy-deficient IECs are much more sensitive to cytokine-induced apoptosis. Therefore we wondered whether TNF blockade would ameliorate the increased IEC death and intestinal pathology observed in colitic poptosis .Figure TNF is a key cytokine in IBD pathology, and treatment strategies targeting TNF signaling are one of the most effective treatment regimens. Despite the proven importance of the cytokine, it is not completely understood how TNF mediates its pathogenic effects and thus how TNF blockade acts to promote disease resolution. Of note, there is considerable heterogeneity among IBD patients in terms of disease manifestations, inflammatory profile and treatment responsiveness. Indeed, although anti-TNF therapies can be very effective, approximately 40% of IBD patients do not respond to anti-TNF treatment and many more become refractory to treatment. TNF blockers are expensive therapeutics and, to date, it is not possible to predict whether a given patient will respond to anti-TNF therapies. Our results suggest that IBD patients harboring risk alleles that sensitize the epithelial barrier to apoptosis may be more likely to respond to anti-TNF therapies, and if confirmed, this could lead to more efficient deployment of such treatments.


MLN4924 therefore inhibits also the βTrCP-dependent activation of the classical and the alternative NFĸB pathway. In this work, we found that a subgroup of multiple myeloma cell lines  and about half of the primary myeloma samples tested are sensitized to TNF-induced cell death by MLN4924. This correlated with MLN4924-mediated inhibition of TNF-induced activation of the classical NFκB pathway and reduced the efficacy of TNF-induced TNFR1 signaling complex formation. Interestingly, binding studies revealed a straightforward correlation between cell surface TNFR1 expression in multiple myeloma cell lines and their sensitivity for MLN4924/TNF-induced cell death. The cell surface expression levels of TNFR1 in the investigated MM cell lines largely correlated with TNFR1 mRNA expression. This suggests that the variable levels of cell surface expression of TNFR1 in myeloma cell lines are decisive for TNF/MLN4924 sensitivity. Indeed, introduction of TNFR1 into TNFR1-negative TNF/MLN4924-resistant KMS-11BM cells, was sufficient to sensitize this cell line for TNF/MLN4924-induced cell death. Thus, MLN4924 might be especially effective in myeloma patients with TNFR1 Several research trials have been directed to treat MM and the current treatment strategies include chemotherapy and stem cell transplantation2. A class of chemotherapeutic drugs which are particular successful in the treatment of MM are proteosome targeting drugs, such as bortezomib and carfilzomib3.Multiple myeloma (MM) is a malignancy of differentiated blood B-cells. This kind of malignancy localizes to the bone marrow leading to osteolysis, bone pain and impaired hematopoiesis5. Activation of NFκB transcription factors occurs via two distinct pathways: the classical and the alternative NFκB pathway5. The classical NFκB pathway depends on activation of the IκB kinase (IKK) complex, e.g., by receptors of the tumor necrosis factor superfamily (TNFRSF) or pattern recognition receptors, IKK-mediated phosphorylation of IκBs, K48 ubiquitination of phospho-IκBs by the E3 ligase β-transducin repeat containing protein (βTrCP) and eventually proteasomal degradation of phosphorylated IκBs. The alternative pathway is primarily triggered by a subset of receptors of the TNFRSF and includes phosphorylation of the IκB-related domain containing NFκB precursor protein p100 by IKK1, phosphorylation of phospho-p100, K48 ubiquitination of phospho-p100, again by the E3 ligase βTrCP, and proteasomal p100 processing to the NFκB subunit p526.The homodimeric and heterodimeric transcription factors of the NFκB family fulfill a variety of functions in different biological processes including immune response, development, cell growth and cell survival. Activation of NFκB transcription factors depends on proteolytic degradation/processing of IκB proteins or proteins containing an IκB-related domain that hinder nuclear translocation of NFκB transcription factors by intermolecular interaction or intramolecular binding, respectively5. TNFR1 is one of the strongest activators of the classical NFκB pathway and can be activated by both soluble and membrane TNF7. On the other hand, the second known TNF receptor, TNFR2, induces mainly the alternative NFκB pathway with only moderate activation of the classical NFκB pathway8. MM is characterized by elevated TNF expression and a deregulated NFκB system10.A variety of stimuli activate the classical NFκB pathway, including physical stressors such as UV light but also receptors of the TNF receptor superfamily or pattern recognition receptors12, MLN4924 acts also as an inhibitor of the two NFĸB pathways by prevention of proteasomal degradation of IκBα and p100 processing16. CRLs and NAE are often particularly active in cancer cells and promote growth, survival and invasion of tumor cells. In line with this, in vitro and in vivo studies revealed anti-tumoral activity of MLN4924 in a variety of cancer models and several clinical phase I studies indicate that MLN4924 is well tolerable18. At the molecular level, the anti-tumoral effects of MLN4924 have been traced back to the inhibition of the proteasomal degradation of various negative regulators of the DNA damage response, the cell cycle and senescence17. Recently, it was proved by us and others that MLN4924 enhances TNF-induced cell death20. As far as proteasome targeting drugs are successfully used to treat MM, an alternative strategy to target the ubiquitin-proteasome pathway in MM might lay in the inhibition of NAE by MLN4924. Therefore, we investigated here the effect of TNF and MLN4924 on the viability of MM cell lines and primary myeloma cells. MM cells were found to be generally TNF resistant but MLN4924 sensitized a subset of myeloma cells for TNFR1-induced cell death. It turned out that cell surface expression of TNFR1 is decisive for TNF/MLN4924 sensitivity.The small molecule MLN4924 (Pevonedistat) inhibits the NEDD8-activating enzyme (NAE) which neddylates the cullin subunit of cullin RING E3 ligases (CRLs). Therefore, this drug acts indirectly as an inhibitor of CRLs, too. Since the βTrCP E3 ligase is a CRLInitially, we compared the cytotoxic effects of MLN4924, carfilzomib and bortezomib on the five human MM cell lines RPMI-8226, MM.1 S, KMS-12BM, AMO-1, and L-363. While bortezomib and carfilzomib induced in nearly all cell lines significant to high cell death within 24 h, MLN4924 had no or only a minor cytotoxic effect Fig. . The lacThe NFκB system has been crucially implicated in the growth and survival of MM cells. The NFκB system is furthermore of overwhelming importance for TNF biology. NFκB signaling not only mediates many of the proinflammatory functions of TNF but also protects most cells from its cell death-inducing activities. Since TNF is typically expressed by immune cells present in the tumor microenvironment of myeloma cells and other cancer entities, we explored the possibility of a synergistic cytotoxic effect of soluble recombinant TNF and MLN4924 on a panel of 10 myeloma cell lines. All multiple myeloma cell lines investigated were resistant against treatment with TNF alone Fig. . In the 7. We found accordingly that only the TNFR1-specific TNF mutant Fc-TNF(32W/86T) but not TNC-scTNF(143N/145R), a highly active TNFR2-specific TNF mutant-based fusion protein8, was able to induce cell death in myeloma cells in the presence of MLN4924  by equilibrium binding studies with RPMI-8226, MM.1S, and KMS-12BM cells  covering 1440 samples furthermore revealed only two mutations in TNFR1, thus far less as for established genes mutated in myeloma, such as TRAF2 and TRAF3  and thus possesses a death domain (DD) in its cytoplasmic part which allows recruitment of defined signaling proteins also carrying a DD28. These DD-containing signaling proteins connect TNFR1 with cytotoxic signaling pathways but also with stimulation of the IKK complex and activation of the classical NFκB pathway. Importantly, the cytotoxic signaling potential of TNFR1 is typically suppressed by varying mechanisms including constitutive and NFκB-dependent expression of anti-apoptotic proteins and modification of signaling proteins involved in cytotoxic TNFR1 signaling, such as RIPK129. The default mode of TNFR1 signaling is therefore typically proinflammatory. TNFR2 is a TNF receptor associated factor (TRAF)-interacting TNFRSF receptor. As such, TNFR2 has no DD but is able to recruit the adapter protein TRAF2 along with its interacting proteins TRAF1 and cellular inhibitor of apoptosis protein-1 (cIAP1) and cIAP228. TNFR2 has thus no own authentic cell death stimulating activity. Noteworthy, TRAF1, TRAF2, cIAP1, and cIAP2 are also indirectly recruited to TNFR1 by the DD-containing signaling proteins TRADD and RIPK1 and fulfill there a dual function. TRAF1/2 and the cIAPs not only promote proinflammatory TNFR1 signaling but also suppress its cytotoxic signaling abilities29.TNF is an extremely pleiotropic cytokine which exerts its cellular effects by stimulation of two receptors of the TNF receptor (TNFR) superfamily (TNFRSF): TNFR1 and TNFR230. TNF is a highly inducible cytokine which is produced by activated immune cells, especially macrophages and T cells32. TNF expression can also be induced in non-immune cells, e.g., fibroblasts, endothelial cells, and epithelia cells by various proinflammatory proteins including TNF itself35. In general the tumor microenvironment contains a high frequency of potentially TNF producing cells and resembles in many aspects chronic inflamed tissues36. Thus, tumor cells are frequently exposed to TNF.While TNFR1 is ubiquitously expressed, TNFR2 expression is limited to certain cell types, particularly myeloid cells and a fraction of T cells and B cells15. MLN4924 therefore acts as an inhibitor of TNF-induced NFκB activation downstream of the activation of the IKK complex. In accordance with the fact that TNFR1-induced NFκB signaling contributes to cell death resistance against TNF, we and others reported that MLN4924 can sensitize cells for the cytotoxic activities of TNF20. MLN4924 is currently under consideration in a variety of clinical trials including phase 1 studies with patients suffering on acute myeloid leukemia, myelodysplastic syndromes, metastatic melanoma, relapsed/refractory MM or lymphoma and solid cancer42. Since TNF has been identified as a factor which is expressed in MM45, we evaluated in this study the different multiple myeloma cell lines and primary myeloma cell samples with respect to their TNF sensitivity in the presence of MLN4924. A subset of multiple myeloma cell lines and also about half of the primary MM samples were found to be sensitized for TNF-induced cell death by MLN4924  and bortezomib from LC Laboratories, Woburn, MA, USA. The SMAC mimetic BV6 was obtained from Syngene .The cell lines KMS-12BM, AMO-1, JJN-3, L-363, OPM-2, RPMI-8226, and U266 were from the German Collection of Microorganisms and Cell Cultures , MM.1S cells were from LGC Standards  and the INA-6 cell line were a kind gift from Martin Gramatzki . MM cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. INA-6 cells were cultured in the presence of 2 ng/ml IL-6. The antibodies used in this study were purchased from Cell Signaling Technology, Beverly, MA, USA , #3130; anti-β-catenin, clone 6B3; anti-IκBα, clone L35A5; anti-phospho-IκBα, clone 14D4; anti-p100/p52, #4882; anti-A20, #5630; anti-TRAF1, #4715; anti-TRAF2, #4724; anti-TNFR1, #3736; anti-IKKβ, clone D30C6; anti-NEDD8, clone 19E3); Enzo Life Sciences, Lörrach, Germany ; BD Biosciences, Germany ; Dunn Labortechnik, Asbach, Germany ; R&D Systems, USA ; Sigma-Aldrich, St. Louis, MO, USA  and Santa Cruz Biotechnology, Germany . zVAD-fmk was obtained from Bachem AG, Weil am Rhein, Germany and nec-1 was obtained from Enzo Life Sciences. Human TNF-α was a kind gift from Prof. Daniela Männel . GpL-TNC-TNF (32W/86T), Fc-TNF(32W/86T) (TNFR1-specific TNF mutant), TNC-scTNF(143N/145R) (TNFR2-specific nonameric TNF mutant)Cells were harvested in ice-cold PBS, were washed twice in PBS, pelleted by centrifugation  and finally total cell lysates were prepared by sonicating cells for 20 s in 4× Laemmli buffer  supplemented with phosphatase inhibitor mixture II (Sigma) and boiling at 95 °C for 5 min. Cell lysates were subjected to vertical SDS-PAGE, and proteins were transferred to nitrocellulose membranes by wet blotting. Blocked membranes were incubated overnight at 4 °C with the primary antibody of interest and the antigen-primary antibody complexes were detected using anti-mouse-HRP  or anti-rabbit-HRP  and the ECL Western blotting detection kit and analysis system from Thermo Fisher Scientific .3 per well). Cells were treated in triplicates with the indicated stimuli and cell viability was determined the next day using the 3--2,5-diphenyltetrazolium bromide (MTT) staining method. In all experiments investigating MLN4924, the compound was added 30 min before cells were treated with TNF etc. Flag-CD95L was oligomerized by incubating the ligand with FLAG-specific mAb M2  30 min before cell stimulation. In experiments using zVAD-fmk/nec-1, cells were treated with zVAD-fmk (50 µM), nec-1 (90 µM) or zVAD-fmk/nec-1 30 min before stimulation with TNF etc. Cell viability values were normalized according to cells without TNF treatment (defined as 100% viable) and cells that have been incubated with a highly cytotoxic cocktail containing sodium azide (defined as 0% viable).Cells were seeded in 96-well tissue culture plates .Equal aliquots of cells were incubated with conventional TNF (50 µg/ml) for 1 h on ice or left untreated. Cells were then incubated pairwise for another hour with increasing concentrations of GpL-TNC-TNF(32W/86T). Then, cells were washed three times with ice cold PBS to remove unbound GpL-TNC-TNF(32W/86T). Cell pellets were finally suspended in 50 µl RPMI culture medium with 0.5% FBS, transferred to a black 96-well plate and assayed for luciferase activity with the Luciferase Assay Kit from New England Biolabs GmbH using a Lumo laminator (Autobio-Diagnostics). Luciferase activities of GpL-TNC-TNF(32W/86T)-treated cells were considered as total binding and luciferase activities of TNF-pretreated/GpL-TNC-TNF(32W/86T)-treated cells were considered as nonspecific binding. Specific binding values were calculated as the difference between total and non-specific binding values and analyzed by nonlinear regression to a one-site specific binding curve with the GraphPad Prism 5 software to obtain the 5) were incubated for 1 h on ice with 200 ng/ml GpL-TNC-TNF(32W/86T) with and without preincubation with TNF (50 µg/ml) for 1 h on ice. Then, cells were washed three times and the experiment was proceeded further as described in the previous section. Cells were counted at the end of each experiment to calculate by help of the measured specific GpL activity of the TNC-GpL-TNF(32W/86T) preparation and the cell bound specific GpL activity, the number of TNFR1 molecules per cell.Cells (5 × 10https://madb.nci.nih.gov/index.shtml).The expression of TNFR1 mRNA in MM cell lines was determined using HG-U133 plus 2.0 microarrays from Affymetrix . Gene expression data was normalized using the MAS5 algorithm and subsequently log2 transformed and annotated to gene symbols using the madb database  overnight and stimulated the next day with Fc-TNF(32W/86T) (2 µg/ml) for 10 min. Cells were harvested and washed twice with ice-cold PBS and lysed for 30 min on ice in 30 mM Tris-HCl (pH 7.5), 1% Triton X-100, 10% glycerol, 120 mM NaCl supplemented with a protease inhibitor  cocktail. Lysates were cleared by centrifugation  and Fc-TNF (32W/86T)-associated TNFR1 complexes were precipitated from the cleared lysates using protein G-agarose beads (30 μl 50% slurry) at 4 °C overnight. Lysates of untreated and MLN4924 alone groups were supplemented with 5 ng Fc-TNF(32W/86T) to serve as a negative control. The next day, protein G beads were washed five times with ice cold lysis puffer and beads were incubated in Laemmli buffer for 15 min at 85 °C to release bound proteins. Protein samples were fractionated and analyzed as described in the Western blotting section.GCTAGCATGGGCCTCTCCACCGTGCC and 5′-CAGGGCGGCCGCCTCATCTGAGAAGACTGGGCG, and cloned into a CMV promotor-based expression cassette of a puromycin resistance-mediating Sleeping Beauty vector. After electroporation into KMS-11 cells (for details see ref. 46) a polyclonal stably transposed culture was established after about two weeks of puromycin selection and TNFR1 cell surface expression assessed. For this, KMS-11 and KMS-11-TNFR1 cells were washed with PBS and incubated with PE-labeled anti-TNFR1 or the corresponding isotype control IgG1 for 30 min at 4 °C. Afterwards, cells were washed three times with ice cold PBS and were then analyzed using a FACSCalibur .The coding sequence of human TNFR1 was amplified and flanked with NheI and NotI sites using primers 5′-GCTG47. Primary cells were not supplemented with IL-6.Bone marrow aspirates from MM patients were obtained at the University Hospital of Würzburg, Department of Internal Medicine II, within the frame of diagnostically indicated aspirations and after informed written consent of the respective patients. Permission was granted by the Ethics Committee of the Medical Faculty, University of Würzburg, Würzburg, Germany . The purification of primary MM cells via microbead selection of CD138-positive cells is described in detail in ref. For a detailed description please see supplemental data