In the few last years, nanosystems have emerged as a potential therapeutic approach to improve the efficacy and selectivity of many drugs. Cyclodextrins (CyDs) and their nanoparticles have been widely investigated as drug delivery systems. The covalent functionalization of CyD polymer nanoparticles with targeting molecules can improve the therapeutic potential of this family of nanosystems. In this study, we investigated cross-linked γ- and β-cyclodextrin polymers as carriers for doxorubicin (ox) and oxaliplatin (Oxa). We also functionalized γ-CyD polymer bearing COOH functionalities with arginine-glycine-aspartic or arginine moieties for targeting the integrin receptors of cancer cells. We tested the Dox and Oxa anti-proliferative activity in the presence of the precursor polymer with COOH functionalities and its derivatives in A549  and HepG2  cell lines. We found that CyD polymers can significantly improve the antiproliferative activity of Dox in HepG2 cell lines only, whereas the cytotoxic activity of Oxa resulted as enhanced in both cell lines. The peptide or amino acid functionalized CyD polymers, loaded with Dox, did not show any additional effect compared to the precursor polymer. Finally, studies of Dox uptake showed that the higher antiproliferative activity of complexes correlates with the higher accumulation of Dox inside the cells. The results show that CyD polymers could be used as carriers for repositioning classical anticancer drugs such as Dox or Oxa to increase their antitumor activity. In the few last years, nanosystems have emerged as a potential therapeutic approach to improve the efficacy and selectivity of drugs ,2. The pd(+)-glucopyranose with the property to act as molecular containers [Cyclodextrins (CyDs) are cyclic oligosaccharides of α-1,4-linked ntainers ,8,9. Thentainers ,11. CyDsntainers , but thentainers . Other fntainers , supramontainers ,18,19,20Polymeric CyD nanoparticles were synthesized through a variety of strategies ,22,23. EIn many cases, epichlorohydrin (EPI) has been used as a suitable cross-linker agent to synthesize CyD polymers .The properties of CyD based NPs were modulated with different approaches to improve their applicability and selectivity in biological systems. The most successful example is CALAA-01 as a gene delivery system. NPs with recognition units were made functional by exploiting adamantane residue affinity for the β-CyD cavity. Adamantane derivatives of transferrin were used as the target unit and successful results were obtained . CyD basActive targeting of the CyD polymers to selectively recognize cancer cells was obtained by covalent attachment of a variety of moieties, such as the luteinizing hormone-releasing hormone analog LHRHa (9 amino acids), folic acids, biotin ,30,31,32In this study, we investigated cross-linked CyD polymers as carriers for doxorubicin (Dox) and oxaliplatin (Oxa), knowing that CyDs can include Dox and Oxa ,36,37,38We tested polymer/drug complexes in lung carcinoma A549 and hepatocarcinoma HepG2 cell lines in comparison with the cross-linked γ-CyD polymer (pγCyD) and anionic β-CyD polymer. A549 and HepG2 cell lines derived from neoplasms placed in the fourth leading causes of death worldwide ,42.EPI cross-linked polymer pγCyDA functionalized with the tripeptide RGD directly or by a PEG4 chain (RGDPEG4) or with Arg methyl ester through a condensation reaction in DMF. The final products were characterized with NMR .1H NMR spectra, signals are broad as typically found for similar systems  were modified with RGD whereas in the case of pγCyDRGD2 only 50% of CyD units (about 14 CyD) were modified.13C NMR spectra showed the alkyl carbons of the RGD moiety and the C=O and C=N carbons at 160–180 ppm  and we investigated only the solubility of Dox (water solubility of free Dox 7.40 × 10−5 M pH 7.4) in the presence of pγCyDA, pβCyDA and pγCyD at pH 7.4. A typical experiment is reported in Oxa has the water solubility of 4 mg/mL (10L-type and have a slope < 1. Complexation efficiency (CE) values of CyD cavity are 0.083 for pγCyDA, 0.082 for pβCyDA and 0.011 pγCyD, suggesting that the presence of carboxylate groups improves the interaction with Dox. While pγCyD only slightly improves the solubility of Dox, the other polymers increase the solubility of Dox very significantly, suggesting a higher affinity for the guest. We did not find a better affinity of pγCyDA compared to pβCyDA. The polymers are EPI cross-linked polymers and an influential role of the network in the Dox interaction can also be proposed.In particular, we reported the Dox solubility versus the CyD unit concentration for a better comparison among the different polymers ,44. The Cell proliferation assay was performed with Dox in the presence of pγCyDA, pβCyDA, pγCyD, pγCyDRGD1, pγCyDRGD2, pγCyDArg, compared to free Dox. At the same time, antiproliferative activity for Oxa was performed only in the presence of pγCyDA, pβCyDA and pγCyD and compared to free Oxa .All polymers alone did not show any toxicity at µM concentration.In A549 cells, Dox-polymer complexes showed a similar or even significantly lower antiproliferative activity (pγCyDA-Dox) in comparison to free Dox.50 value as compared to Dox alone.Conversely, in HepG2 cells pγCyDA-Dox, pβCyDA-Dox and pgCyD-Dox showed always a noteworthy and significant decrease of ICWhen Oxa was administered to A549 and HepG2 cells in the presence of pγCyDA, pβCyDA and pγCyD its antiproliferative activity was always significantly improved as compared to the treatment with Oxa alone .In order to study the possible effect of functionalization of cross-linked polymers, we synthesized RGD and Arg functionalized polymers and tested the new complexes pγCyDRGD1-Dox, pγCyDARGD2-Dox, and pγCyDArg-Dox .These RGD functionalized polymers showed an antiproliferative activity similar to that found for the Dox complex with pγCyDA , both inIn particular, the fact that pγCyDRGD2-Dox showed a significantly lower antiproliferative effect than free Dox may be due to the presence of PEG chain that can modify the features of the system.This result is of interest because the effect of the polymers on the Dox toxicity is generally significant, while in many cases reported elsewhere the presence of NPs reduced the cytotoxicity of the drug. The entrapment into NPs may reduce the ability of the drug to cross the cell membrane. In this case, we found that anionic polymers can include Dox better than the pCyD. This data can explain the decreased activity in A549 cells. On the contrary, in the case of HepG2 cells, pγCyDA is the most effective system. Probably the type of transporters of the HepG2 cells may explain this behavior .We cytofluorimetrically evaluated the drug uptake in A549 and HepG2 cells after exposure to equal concentrations of Dox/CyD complexes and Dox alone to prove that the difference of anti-proliferative activity of Dox/CyD polymers compared to free Dox was determined by a differential Dox accumulation into target cells.Our results clearly show that Dox accumulation was strictly dependent on the type of CyD-polymer utilized for drug delivery although with evident differences between A549 and HepG2 cells .p < 0.05). These data are inversely correlated with the analysis of the antiproliferative activity of such complexes , which always had IC50 values lower than that of the parent compound alone , anionic γCyD polymer  were purchased from Cyclolab. 1-(3-Dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride , N-Hydroxy succinimide ; doxorubicin hydrochloride (Carbosynth), oxaliplatin , arginine methyl ester dihydrochloride  were used. Free base ArgOCH3 was obtained using a DEAE (OH- form) anionic column.Sephadex G-15 and sephadex-DEAE A-25 were used for column chromatography.Thin-layer chromatography (TLC) was carried out on silica gel plates. Carbohydrate derivatives were detected on TLC by UV, anisaldehyde test or ninhydrin.1H NMR spectra were recorded at 25 °C with a Varian UNITY PLUS-500 spectrometer at 499.9 and 125.7 MHz respectively, using standard pulse programs from the Varian library. Two-dimensional experiments  were performed using 1K data points, 256 increments. The mixing times for NOESY were 200–400 ms. Spectra were referred to the solvent signal.UV-Vis spectra were recorded with VersaWave microvolume UV/Vis spectrophotometer.Dynamic light scattering (DLS) measurements were performed at 25 °C with Zetasizer Nano ZS  operating at 633 nm (He–Ne laser). The mean hydrodynamic diameter (d) of the NPs was calculated from intensity measurement after averaging the five measurements. The samples (1 mg/mL) were diluted in phosphate buffer (pH = 7.4) prepared in ultrapure filtered water (0.2 µm filter). The Z average values of commercial polymers were also measured: pγCyD 35 ± 3 nm, pγCDA 25 ± 2 nm, pβCDA 53 ± 5 nm.v/v/v). Crude peptides were recovered by precipitation with freshly distilled diethyl ether.The RGD and PEG4RGD peptide were carried out on a Novabiochem TGR resin  using the Fmoc chemistry method. All Fmoc amino acids were introduced according to the Dic/oxyma activation method . The 15-1H NMR  δ ppm): 4.00–3.96 ; 4.00–3.96 ; 3.58 ; 3.16–3.10 ; 2.71 ; 1.84 , 1.64–1.52 .RGD: 1H NMR  δ (ppm): 4.54–4.48 ; 4.22–4.16 ; 3.81 ; 3.69–3.46 ; 3.07 ; δ 2.72–2.58 ; 2.50–2.40 ; 1.79–1.45 .RGD-PEG4: pγCyDA (226 mg) was dissolved in DMF (10 mL). NHS (22.7 mg) and EDC (13.7 mg), previously dissolved in 500 μL of DMF, were added. A DMF solution of RGD  was added drop by drop to the solution of pγCyDA during 12 h. The reaction mixture was stirred for 3 days at room temperature. TLC showed the formation of the new products. The product was isolated with a Sephadex G-15 column, using water as eluent.The appropriate fractions were collected and analyzed by NMR. Acetone was added to the solid products and the precipitate was collected by centrifugation.1H NMR:  δ (ppm): 1.92–1.51 ; 2.64–2.55 ; 3.21–2.99 ; 4.16–3.18 ; 5.70–4.93 .Dimension : 27 ± 3 nm.The synthesis was carried out as reported for pγCyDRGD1, using RGD-PEG4.After purification with Sephadex G-15, three fractions were collected and analyzed by NMR. The fractions were purified again with an anionic DEAE column chromatography.The sample was precipitated by acetone, centrifuged and dried.1H NMR:  δ (ppm): 1.86–1.51 ; 2.6–2.55 ; 2.55–2.45 ; 3.16–3.10 ; 4.10–3.19 ; 4.26 ; 4.51–4.46 ; 5.75–4.83 .13C NMR  δ (ppm): 24.5 (b RGD); δ 24.9 (c RGD); 35.8 (h RGD); 40.7 (a RGD); 42.6 (f RGD); 60.5 (PEG4), 62.8 (PEG4); ; 79–81 ; 102–104 ; 156.7 ; δ 171.0 ; 174.5 (CONH); 176.2 177.4–177.8 (CONH); 178.0 (CONH2).Dimension : 30 ± 3 nm.3) was added under stirring. After 4 days the solution was purified via a Sephadex G-15 column and fractions obtained were analyzed through TLC chromatography.pγCyDA (82.60 mg) was dissolved in 5 mL of DMF and NHS (5.86 mg in 500 μL) and EDC (9.82 mg in 500 μL) were added. Arginine methyl ester hydrochloride  δ 5.55–4.85 ; 4.31–3.12 ; 3.10 ; 1.86–1.4 .Dimension : 26 ± 3 nm.−1 L cm−1). The CE (complexation efficient) was calculated from the slope of the straight line obtained. CE = Slope/(1 − Slope).Dox hydrochloride  was added to 0.200 mL of 8 different concentration solutions of the CyD polymers in phosphate buffer  as reported elsewhere . The susHuman cell lines A549  and HepG2  were plated 180 µL into flat-bottomed 96-well microtiter plates at 1200/mL and 2000/mL, respectively. After 6–8 h, cells were treated with 20 µL containing five concentrations of Dox or Oxa alone or loaded in CyD polymers diluted in normal saline solution. Plates were then processed as described elsewhere . Dox wit50) were calculated based on the analysis of the concentration–response curves. Each experiment was repeated 4–11 times.The concentrations inhibiting 50% cell growth  using 488 nm excitation and 655–730 nm emission filters. Data were analyzed with FowJo 10.7.2 software Values were normalized using the Staining Index (SI):The Mann–Whitney and the Wilcoxon signed-rank tests and the Pearson correlation coefficient were used for the statistical analysis of data.


Phe accumulation and increased antibiotic persistence. This increase in antibiotic persistence was most pronounced (65-fold higher) when the relA-encoded tRNA-dependent stringent response was inactivated. The increase in persistence with elevated quality control correlated with ∼2-fold increases in the levels of the RNase MazF and the NTPase MazG and a 3-fold reduction in cellular NTP pools. These data reveal a mechanism for persister formation independent of the stringent response where reduced translation capacity, as indicated by reduced levels of aminoacylated tRNA, is accompanied by active reduction of cellular NTP pools which in turn triggers antibiotic persistence.Bacterial antibiotic persistence occurs when bacteria are treated with an antibiotic and the majority of the population rapidly dies off, but a small subpopulation enters into a dormant, persistent state and evades death. Diverse pathways leading to nucleoside triphosphate (NTP) depletion and restricted translation have been implicated in persistence, suggesting alternative redundant routes may exist to initiate persister formation. To investigate the molecular mechanism of one such pathway, functional variants of an essential component of translation  were used to study the effects of quality control on antibiotic persistence. Upon amino acid limitation, elevated PheRS quality control led to significant decreases in aminoacylated tRNA Bacterial persisters are a subpopulation of dormant microbial cells that are not actively growing or dividing, which causes them to be tolerant to bactericidal antibiotics . Once thEscherichia coli ΔrelA ΔspoT background that is unable to synthesize ppGpp  PheRS is 22, and for αA294G PheRS, it is 1.8, confirming that editing is required to prevent misacylated m-Tyr-tRNAPhe accumulation and that αA294G PheRS can only minimally discriminate between Phe and m-Tyr. In contrast, the specificity of Phe-to-m-Tyr for αA294S PheRS is 370, indicating that this mutant is not able to efficiently activate m-Tyr for aminoacylation. The same trend follows for the specificity of Phe-to-Tyr: αA294G PheRS has a reduced substrate specificity compared to wild-type PheRS, and αA294S PheRS has an increased specificity.Along with an kgrounds –31. The mination . These rvariants . The sperelA+ and ΔrelAE. coli backgrounds  in both backgrounds were grown to early log phase and then treated with 100 μg/ml ampicillin for 3 h; each hour, an aliquot was taken out, washed with sterile phosphate-buffered saline (PBS), plated on LB plates, and incubated at 37°C overnight to calculate CFU. These assays were performed in two different types of media. The first, medium A, is the control medium which is a supplemented M9-based minimal medium that contains 40 μg/ml of all 20 proteogenic amino acids. The second, medium B, is the starvation medium which is also a supplemented M9-based minimal medium that contains 40 μg/ml of 18 proteogenic amino acids, 10 μg/ml Tyr, 40 μg/ml m-Tyr, and no Phe. The total amounts of persisters for wild-type PheRS, editing-deficient PheRS, and the two different active site PheRS mutant strains in both E. coli backgrounds were calculated at the endpoint after 3 h of exposure to ampicillin  assays were performed in both MG1655 picillin . There w10.1128/mBio.01132-21.2FIG S1E. coli PheRS strains in both a relA+ and ΔrelA background. Cultures were grown to early log phase and then exposed to 100 μg/ml ampicillin for 3 h. Cultures were taken out every hour, washed in sterile PBS, and plated to calculate CFU. (A) WT pheS/pheT; (B) editing-deficient PheRS, pheT G318W; (C) active site mutant PheRS, pheS A294G; (D) active site mutant pheS A294S. In each panel, MDK assay was performed in relA+E. coli (black lines), ΔrelAE. coli (colored lines), medium A (solid lines), and medium B (dashed lines). Medium A is a supplemented M9 minimal medium that contains 40 μg/ml of all 20 proteogenic amino acids, and medium B is a supplemented M9 minimal medium with 40 μg/ml of 18 proteogenic amino acids, 10 μg/ml Tyr, 40 μg/ml m-Tyr, and no Phe. Error bars represent standard deviation from 3 biological replicates. Download FIG S1, PDF file, 0.1 MB.Minimum duration of killing (MDK) assays for different Copyright © 2021 Wood et al.2021Wood et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the pheT G318W/pheS A294G double mutant was made in both the E. coli MG1655 relA+ and ΔrelA backgrounds. These strains would be able to synthesize, but not edit, m-Tyr-tRNAPhe and Tyr-tRNAPhe and therefore would generate high levels of misacylated tRNAPhe in the cell. The pheT G318W/pheS A294G double mutation allowed normal growth compared to the wild-type PheRS in medium A in both the relA+ and ΔrelA backgrounds. However, we were not able to perform persister assays on the pheT G318W/pheS A294G/ΔrelA mutant because the cells showed a substantial growth defect in medium B  and double mutant PheRS, pheS A294G/pheT G318W (dashed lines), in either medium A (green) or medium B (blue) or with ΔrelAE. coli with wild-type PheRS (solid line) and double mutant PheRS (dashed lines) in either medium A (purple) or medium B (orange). Medium A is a supplemented M9 minimal medium that contains 40 μg/ml of all 20 proteogenic amino acids, and medium B is a supplemented M9 minimal medium with 40 μg/ml of 18 proteogenic amino acids, 10 μg/ml Tyr, 40 μg/ml m-Tyr, and no Phe. Error bars represent standard deviation from 3 biological replicates. Download FIG S2, PDF file, 0.1 MB.Growth curves comparing wild-type PheRS and double mutant Copyright © 2021 Wood et al.2021Wood et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Phe, assays were performed to quantify tRNAPhe aminoacylation in all four PheRS strains in both relA+ and ΔrelAE. coli backgrounds grown in either medium A or medium B . There was a general trend of having a lower intracellular nucleotide concentration in the strains that showed elevated levels of persistence  relA+pheS A294S versus relA+ wild-type pheS. (B) ΔrelA wild-type pheS versus relA+ wild-type pheS. (C) ΔrelA pheS A294S versus relA+pheS A294S. (D) ΔrelA pheS A294S versus ΔrelA wild-type pheS. (E) ΔrelA pheS A294S versus relA+ wild-type pheS. Download FIG S3, PDF file, 0.1 MB.Disruption of proteome homeostasis represented by volcano plots. Total proteome analysis was performed on wild-type Copyright © 2021 Wood et al.2021Wood et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01132-21.6TABLE S1relA WT pheS/pheT versus relA+ WT pheS/pheT grown in medium B. Download Table S1, DOCX file, 1.8 MB.Differentially expressed proteins in ΔCopyright © 2021 Wood et al.2021Wood et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01132-21.7TABLE S2relApheS A294S versus relA+ WT pheS/pheT grown in medium B. Download Table S2, DOCX file, 1.8 MB.Differentially expressed proteins in ΔCopyright © 2021 Wood et al.2021Wood et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01132-21.8DATA SET S1pheS and pheS A294S in either MG1655 relA+ and ΔrelA backgrounds grown in medium B. Over- and underrepresented proteins are noted. Download Data Set S1, XLSX file, 1.2 MB.Total proteome analysis. Changes in protein abundance were monitored for wild-type Copyright © 2021 Wood et al.2021Wood et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the pheS A294S strains  Reaction of active site mutant αA294G PheRS, which has decreased specificity for m-Tyr and Tyr compared to wild-type PheRS. The noncognate amino acid will be efficiently aminoacylated onto tRNAPhe, which will be moved to the editing site where hydrolysis of the misacylated tRNAPhe will occur. Deacylated tRNAPhe and noncognate amino acid will be released from PheRS. (B) Reaction of active site mutant αA294S PheRS, which has increased specificity for m-Tyr and Tyr compared to wild-type PheRS. The noncognate amino acid will not bind and therefore will not be aminoacylated onto tRNAPhe. Download FIG S4, PDF file, 0.1 MB.Molecular model of increased deacylated tRNACopyright © 2021 Wood et al.2021Wood et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the pheT G318W, pheS A294G, and pheS A294S, were made on the chromosome of E. coli MG1655 and the corresponding ΔrelA strain. Intriguingly, there was a reduction in persistence in the pheT G318W ΔrelA strain grown under amino acid limitation ppGpp and the cells would enter into a persister state ppGpp that MazG would be degrading is being produced from SpoT. It is also possible that MazG might be degrading NTPs to prevent the synthesis of (p)ppGpp, since it was observed from the metabolomics that ATP and GTP concentrations were reduced in these strains. Furthermore, to ensure that the increase in the expression levels of MazF and MazG was not an artifact of transcriptional read-through from the disruption of relA using a kanamycin cassette, sequencing of this operon was performed (data not shown). We confirmed that both the 5′ untranslated region (UTR) and 3′ end of relA are intact and that the 5′ region of mazEF, which contains both of its promoters, was not disrupted by the kanamycin insertion into the relA gene. Both of these promoters are required for the autoregulation of mazEF, and when MazE is degraded, MazF is released as a toxin  background or a ΔrelAE. coli background   reached 0.1 to 0.2, λ-red expression was induced with 0.2% arabinose. Once the culture reached an OD600 of 0.4 to 0.6, 2 μM single-stranded DNA (ssDNA) recombineering oligonucleotide that contained the desired mutation was transformed by electroporation. The pheS A294G oligonucleotide sequence was 5′-AACGCAACATAGTCAGACGCTCCATCCCCATCCCAAACCCAAAACCGCTATAAACTTCGGGATCAATGCCAACGTTACGC-3′, and the pheS A294S oligonucleotide sequence was 5′-AACGCAACATAGTCAGACGCTCCATCCCCATCCCAAAGCTAAAACCGCTATAAACTTCGGGATCAATGCCAACGTTACGC-3′. Both recombineering oligonucleotides had four phosphorothioate bonds at the 5′ end of the oligonucleotide. Recombineering occurred at 30°C for 2 h while shaking. Counterselection is achieved by induction of Cas9 with anhydrotetracycline. pKDsgRNA-pheS and pCas9Cr4 curing was performed exactly as described previously (CTCGCAGAACTGGTTGCCAG-3′ and reverse primer 5′-CACGCAGTTTGTCAGCGTTCG-3′. Clones were confirmed by DNA sequencing using forward primer 5′-CTGATTGTTGATACCAACATC-3′. E. coli XL1-Blue/pQE31-FRS (producing His6-tagged wild-type PheRS) and E. coli XL1-Blue/pQE31-pheSA294G (producing His6-tagged αA294G PheRS) were previously constructed (pheS gene of pQE31-FRS by PCR-based site-directed mutagenesis using two self-complementary primers (5′-ACTCTGGTTTCAGCTTCGGGATGG-3′) to generate E. coli XL1-Blue/pQE31-pheSA294S (producing His6-tagged αA294S PheRS). The mutation was confirmed by DNA sequencing. Protein expression and purification were performed essentially as described previously  , 31. Bristructed . A pointeviously .i exchange to determine steady-state kinetics of amino acid activation are in Detailed description of the methods for ATP/PP10.1128/mBio.01132-21.1TEXT S1Text S1, DOCX file, 0.02 MB.Methods and Materials. Download Copyright © 2021 Wood et al.2021Wood et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 4, 0.2 mM CaCl2, 2 μg/ml thiamine, 1 μg/ml FeSO4 solution, 80 μg/ml all proteogenic amino acids except for phenylalanine and tyrosine. For the persister assays, two different media were used, medium A and medium B. Medium A contained 1× supplemented M9 minimal medium, 40 μg/ml phenylalanine, and 40 μg/ml tyrosine. Medium B contained 1× supplemented M9 minimal medium, 10 μg/ml tyrosine, and 40 μg/ml m-tyrosine .Lysogeny broth (LB) was made with 0.5% NaCl, 0.5% yeast extract, and 1% tryptone. A supplemented M9 minimal medium that was used for persister assays was developed based on a previous protocol . A 2× su600 = 0.2 to 0.3). At this point 1 ml of culture was taken out, serially diluted, and plated on LB plates to count CFU; 100 μg/ml ampicillin was added to the remaining culture; and growth continued at 37°C with shaking. Every hour for 3 h, 1 ml of culture was removed, washed in sterile phosphate-buffered saline (PBS) twice to remove the antibiotic, serially diluted, and plated on LB plates. To calculate persisters, the CFU of survivors after ampicillin treatment was divided by the CFU of the culture before ampicillin treatment.MDK persister assays were performed as described in several studies , 14, 54.Purification of total tRNA, acid/urea gel electrophoresis, and Northern blotting methods are described in detail in E. coli cells as described previously  with a spray voltage of 3,500 V in the negative ion mode, ion vaporizer at 50°C, and ion transfer tube at 350°C. Analysis and quantification were performed using Xcalibur data acquisition and interpretation software. Intracellular concentrations were calculated assuming an OD600 of 1.0 equals 8 × 108 cells and that the volume of one E. coli cell growing in exponential phase equals 1 × 10−15 liter  solution, resulting in a sample pH of <3. Remaining ALS reagent was cleaved for 15 min at room temperature. The sample (∼30 μg protein) was desalted by reverse-phase cleanup using C18 UltraMicroSpin columns. The desalted peptides were dried at room temperature in a rotary vacuum centrifuge and reconstituted in 20 μl 70% formic acid-0.1% TFA (3:8 [vol/vol]) for peptide quantitation by UV280. The sample was diluted to a final concentration of 0.4 μg/μl, and 5 μl (2 μg) was injected for LC-MS/MS analysis.Twenty-milliliter cultures were inoculated to a starting OD at 600 nm of 0.01 in either medium A or medium B using an overnight culture to stationary phase. After reaching mid-log, cells were chilled on ice and pelleted by centrifugation for 2 min at 8,000 rpm. The resulting pellet was frozen at −80°C for downstream processing. For cell lysis and protein digest, cell pellets were thawed on ice and 2 μl of cell pellet was transferred to a microcentrifuge tube containing 40 μl of lysis buffer . Cells were lysed by vortex for 30 s, and disulfide bonds were reduced by incubating the reaction mixture for 30 min at 55°C. The reaction was briefly quenched on ice, and 16 μl of a 60 mM iodoacetamide solution was added. Alkylation of cysteines proceeded for 30 min in the dark. Excess iodoacetamide was quenched with 14 μl of a 25 mM DTT solution, and the sample was then diluted with 330 μl of 183 mM Tris-HCl buffer (pH 8.0) supplemented with 2 mM CaCl18-AQ (Dr. Maisch) using methanol as the packing solvent. Peptide separation was achieved using mixtures of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) with a 90-min gradient of either 0/1, 2/7, 60/24, 65/48, 70/80, 75/80, 80/1, or 90/1 . Gradient was performed with a flow rate of 250 nl/min. At least one blank injection (5 μl 2% B) was performed between samples to eliminate peptide carryover on the analytical column. One hundred femtomoles of trypsin-digested bovine serum albumin (BSA) or 100 ng trypsin-digested wild-type K-12 MG1655 E. coli proteins was run periodically between samples as quality control standards.The mass spectrometer was operated with the following parameters: (MS1) 70,000 resolution, 3e6 automatic gain control (AGC) target, 300 to 1,700 m/z scan range; (data-dependent MS2) 17,500 resolution, 1e6 AGC target, top 10 mode, 1.6 m/z isolation window, 27 normalized collision energy, 90-s dynamic exclusion, unassigned, and +1 charge exclusion. Data were searched using MaxQuant version 1.6.10.43 with deamidation (NQ), oxidation (M), and phospho (STY) as variable modifications and carbamidomethyl (C) as a fixed modification with up to 3 missed cleavages, 5-amino-acid (aa) minimum length, and 1% false-discovery rate (FDR) against a UniProt E. coli database. Search results were analyzed with Perseus version 1.6.2.2.LC-MS/MS was performed using an Acquity UPLC M-class (Waters) and Q Exactive Plus mass spectrometer. The analytical column employed was a 65-cm-long, 75-μm-internal-diameter PicoFrit column (New Objective) packed in-house to a length of 50 cm with 1.9-μm ReproSil-Pur 120-Å C


Pathologic tau modifications are characteristic of Alzheimer's disease and related dementias, but mechanisms of tau toxicity continue to be debated. Inherited mutations in tau cause early onset frontotemporal lobar dementias (FTLD-tau) and are commonly used to model mechanisms of tau toxicity in tauopathies. Previous work in the isolated squid axoplasm model demonstrated that several pathogenic forms of tau inhibit axonal transport through a mechanism involving activation of protein phosphatase 1 (PP1). Here, we determined that P301L and R5L FTLD mutant tau proteins elicit a toxic effect on axonal transport as monomeric proteins. We evaluated interactions of wild-type or mutant tau with specific PP1 isoforms  to examine how the interaction contributes to this toxic effect using primary rat hippocampal neurons from both sexes. Pull-down and bioluminescence resonance energy transfer experiments revealed selective interactions of wild-type tau with PP1α and PP1γ isoforms, but not PP1β, which were significantly increased by the P301L tau mutation. The results from proximity ligation assays confirmed the interaction in primary hippocampal neurons. Moreover, expression of FTLD-linked mutant tau in these neurons enhanced levels of active PP1, also increasing the pausing frequency of fluorescently labeled vesicles in both anterograde and retrograde directions. Knockdown of PP1γ, but not PP1α, rescued the cargo-pausing effects of P301L and R5L tau, a result replicated by deleting a phosphatase-activating domain in the amino terminus of P301L tau. These findings support a model of tau toxicity involving aberrant activation of a specific PP1γ-dependent pathway that disrupts axonal transport in neurons.SIGNIFICANCE STATEMENT Tau pathology is closely associated with neurodegeneration in Alzheimer's disease and other tauopathies, but the toxic mechanisms remain a debated topic. We previously proposed that pathologic tau forms induce dysfunction and degeneration through aberrant activation of a PP1-dependent pathway that disrupts axonal transport. Here, we show that tau directly interacts with specific PP1 isoforms, increasing levels of active PP1. Pathogenic tau mutations enhance this interaction, further increasing active PP1 levels and impairing axonal transport in isolated squid axoplasm and primary hippocampal neurons. Mutant-tau-mediated impairment of axonal transport was mediated by PP1γ and a phosphatase-activating domain located at the amino terminus of tau. This work has important implications for understanding and potentially mitigating tau-mediated neurotoxicity in tauopathies. MAPT (microtubule-associated protein tau) gene encoding tau induce early onset neurodegeneration classified within frontotemporal lobar dementias , a complex set of microtubule-based intracellular trafficking events involving bidirectional movement of organelles along axons, is one cellular process affected by pathogenic tau. Using squid axoplasm preparations, a well-established experimental model to study FAT, we showed that physiological, or even supraphysiological, levels of normal human tau monomers did not negatively affect FAT . In contIn this study, we test the hypothesis that two FTLD-tau mutations disrupt FAT via a PP1-dependent mechanism in mammalian neurons. Using protein–protein interaction assays we found that tau directly interacts with selected PP1 isoforms, and the disease-causing P301L mutation enhances such interactions. Measuring a regulatory phosphorylation site in PP1, we also show that tau increases active PP1 levels, an effect enhanced by FTLD-tau mutations. We demonstrate that both P301L and R5L mutant tau, but not wild-type (WT) tau, impair FAT using isolated squid axoplasm and cultured neurons as complementary experimental systems. In mammalian neurons, the tau mutants impaired FAT by increasing cargo pause frequency in a PP1γ isoform-dependent and PAD-dependent fashion. These findings establish a specific molecular mechanism by which two disease-causing tau forms similarly induce FAT impairments in mammalian neurons.Escherichia coli using pT7C backbones and IPTG induction, as previously described  in both WT- and FTLD-related P301L and R5L mutant forms . All tauescribed . Brieflytrations .Doryteuthis pealeii  was used to measure the effects of tau monomers on FAT, as previously described (m): 175 potassium aspartate, 65 taurine, 35 betaine, 25 glycine, 10 HEPES, 6.5 MgCl2, 5 EGTA, 1.5 CaCl2, 0.5 glucose, and 5 adenosine triphosphate, pH 7.2, to a final concentration of 2 μm and perfused into isolated axoplasm. Anterograde and retrograde FAT rates were alternately obtained by matching the calibrated cursors to the bulk flow of membrane-bounded organelles  for 50 min after perfusion, as described previously  was attached to the N-terminal end of each of the PP1α, PP1β, and PP1γ1 isoforms , 5% FBS, 1% penicillin/streptomycin. Cells were not used beyond passage 30.HEK293T cells (ATCC) were grown at 37°C and 5% COg for 2 min to settle resin between each wash. Cells were lysed in 150 μl of lysis buffer , then homogenized 20× with disposable polypropylene dounce homogenization pestles (Sigma-Aldrich). The samples were centrifuged at 14,000 × g for 5 min at 4°C, and the supernatant was transferred to a new tube containing 350 μl of TBS . Next, 450 μl of each sample lysate/TBS mix was transferred to the HaloLink resin beads, and the remainder of the lysates were frozen as preincubation lysate samples. The samples were incubated for 1 h on a rotating mixer to allow covalent binding of the HaloTagged PP1 or HaloTag-only as control. After incubation, samples were centrifuged at 800 × g for 2 min, and the supernatants were saved as post-pull-down lysate samples. The beads were washed 4× in 800 μl of wash buffer with the final wash step including a 5 min incubation on the rotating mixer. After removing the wash buffer, the beads were incubated in 50 µl of 2× sample buffer  for 30 min at 1000 rpm and 30°C in an Eppendorf ThermoMixer C. After a centrifugation at 800 × g for 2 min, the supernatants were collected as the pull-down sample.Three hundred thousand HEK293T cells/well were plated into a 12-well polystyrene plate. After 24 h, the cells were transfected with 500 ng each of pCMV tau-NanoLuc  and pCMV Halo-PP1  or a pCMV Halo control  using Lipofectamine 2000 and allowed to express proteins for 18 h before collection. Immediately before cell lysis, 50 μl of HaloLink resin  was aliquoted for each sample. The beads were washed 3× in 500 μl of wash buffer  with centrifugation steps of 800 × m Tris, pH 6.8, 2% SDS, 6% glycerol, 1% β-mercaptoethanol, 0.002% bromophenol blue). These samples and the pull-down samples (eluted in 2× sample buffer) were heated at 99°C for 5 min. Fifteen microliters of each of the lysate samples and 25 μl of the pull-down samples were separated using Criterion TGX Precast 4–20% gels (Bio-Rad) in SDS-PAGE. The proteins were then transferred to BioTrace NT nitrocellulose transfer membrane  at 400 mA for 50 min. The membranes were blocked in 2% milk/TBS for 1 h at room temperature and then probed overnight at 4°C with primary antibodies. All antibodies were diluted in 2% milk/TBS. The R1 polyclonal rabbit antibody  was used to detect tau (AB_2688011) was used to detect HaloTag proteins. Blots were rinsed with 0.1% Tween 20/TBS (TBST) and then incubated with appropriate secondary antibodies diluted 1:20,000 in milk/TBS . The blots were imaged using a Li-Cor Odyssey infrared imaging system, and Li-Cor ImageStudioLite 5.2 software was used to quantify immunoreactivity of bands. The bulk of Halo-PP1 or Halo tag alone covalently binds to the HaloLink resin and does not elute from the beads. Therefore, relative tau levels in pull-down samples were calculated by normalizing tau signal from the elution fraction to the change in Halo signal [tau elution signal/].For immunoblots, 12.5 μl of preincubation and postincubation lysate samples were added to 2.5 μl of 6× sample buffer  technique using a donor and acceptor tags was used to demonstrate protein–protein interaction . The DNAg for 2 min and resuspended in 1 ml of OptiMEM . Aliquots of each sample were diluted in trypan blue (Bio-Rad) for cell counting. The cells were diluted to a volume of 660 μl at 200 cells/μl in OptiMEM . The cells were split into two samples. One received 0.33 μl of 618 ligand (Promega), and the other received 0.33 μl of DMSO as a control. The 618 ligand attaches to the HaloTag and acts as the acceptor fluorophore, whereas the DMSO control contains no fluorophore. Six wells of each transfection condition were plated onto a 96-well, white-wall, clear-bottom plate  at 20,000 cells and 100 μl/well then incubated for 18–20 h. A 5× stock solution of Nano-Glo substrate (Promega) was prepared in OptiMEM . Twenty-five microliters of the Nano-Glo substrate stock was then added to each well and immediately placed in the BioTek Synergy NEO HTS plate reader equipped with filtered luminescence (BioTek). After shaking for 30 s, the luminescence values were read using a 410/80 bandpass filter  and 610 nm long-pass filter .After 18 h, the cells were detached from the plate on addition of 500 μl of 0.5% trypsin and a brief incubation at 37°C. After dissociation, the trypsin was quenched with 1 ml of cell culture media. The cells were centrifuged at 200 × The mean corrected nBRET ratios were calculated according to the provided manufacturer protocol. The raw milliBRET unit (mBU) ratio was calculated by dividing the acceptor luminescence values by the donor luminescence values and then multiplying by 1000. These values were then corrected by subtracting the control milliBRET ratios (with DMSO) from the experimental milliBRET ratios (with 618 ligand). The mean and SD of these values were plotted versus the transfection DNA ratios and fit to a hyperbolic curve using GraphPad Prism.ad libitum. Procedures were conducted in compliance with federal, state, and institutional guidelines and approved by the Michigan State University Institutional Animal Care and Use Committee.Timed-pregnant Sprague Dawley rats (Harlan Laboratories) were used to obtain embryonic d 18 (E18) fetuses (both sexes) for culturing primary hippocampal neurons, as previously described . Rats weg at 4°C) and then resuspended and plated in antibiotic-free neurobasal media (Invitrogen) supplemented with 1% GlutaMAX (Invitrogen) and 2% B-27 (Invitrogen).Briefly, hippocampi were dissected from the rat embryos, cut into small pieces, and placed in ice-cold calcium- and magnesium-free buffer . The tissue was washed 4× with CMF and incubated in 0.125% trypsin for 15 min at 37°C and washed again 2× with CMF then suspended in trypsin-inactivation solution , 50 μg/ml DNase I (Worthington), and 20% newborn calf serum (Invitrogen). Cells were dissociated through trituration using progressively smaller diameter syringes, pelleted over FBS  and 1.2 ml sterile deionized water. Then 400 μl of 2.5 m CaCl2 was mixed into the solution and incubated for 5 min at RT followed by a dropwise addition of 4 ml of 2× HBS containing the following (in mm): 281 NaCl, 100 HEPES, and 1.5 Na2HPO4, pH 7.12, into the gently vortexed solution. The transfection solution was then evenly distributed throughout the plates at 2 ml/dish and incubated overnight. The media was then changed to viral cell culture media . The supernatants were collected on days 3 and 4 post-transfection by centrifuging at 675 × g for 5 min, then filtering through 0.45 μm filters. A 2 ml 20% sucrose bed was laid below the clarified media in ultracentrifuge tubes then spun in a swinging bucket rotor at 82,700 × g for 2 h at 4°C. The supernatant was removed, and the pellet was resuspended in 500 μl of PBS then aliquoted and frozen at −80°C after the second collection. Lentiviral titers were determined using a Lenti X P24 Rapid Titration Elisa Kit (TaKaRa Bio) according to manufacturer protocol, and similar levels of tau expression were confirmed via immunoblotting before use.Lentiviral stocks were prepared to individually express WT tau, P301L tau, R5L tau, and GFP in primary neuron cultures. DNA constructs including a pHEF VSV-G envelope vector, a pNHP packaging vector , and a pin vitro (DIV) 4 to express WT tau, P301L tau, or R5L tau. Additional control wells were left untreated with virus. After 4 d of incubation the cells were fixed with a 20 min treatment of 4% paraformaldehyde in cytoskeleton buffer containing the following (in mm): 10 MES, 138 KCl, 3 MgCl2, and 4 EGTA, pH 6.1, followed by three 5 min washes in TBS. The cells were permeabilized and incubated in blocking buffer  for 1 h at room temperature. Neurons expressing each tau construct and the untreated cells were then incubated with Tau7 antibody  or a PP1γ-specific antibody  at 1:500 dilutions in 2% goat serum/TBS overnight at 4°C. Neurons expressing WT tau and untreated cells were incubated with each antibody individually or no primary antibodies for proximity ligation assays (PLAs) method controls. The Duolink PLA Control Kit (Sigma-Aldrich) was used to identify an association between tau and PP1 in the neurons. The cells were washed 3× 5 min at room temperature Buffer A then incubated with anti-mouse PLUS and anti-rabbit MINUS secondary probes (1:5 dilution each) in 2% goat serum for 1 h at 37°C. After 3 additional 5 min washes in buffer A, the neurons were incubated in ligase (1:40 dilution) in 1× ligation buffer for 30 min at 37°C and washed 3× in Buffer A again. Next, the neurons were incubated with polymerase (1:80 dilution) in 1× amplification buffer for 100 min at 37°C followed by incubation in blocking buffer for 1 h at room temperature. Then cells were incubated overnight at 4°C with biotinylated Tau12 antibody  at a 1:1000 dilution and Alexa Fluor goat-anti mouse IgG2a 647 antibody  in 2% goat serum for 1 h at RT then washed again 3× 5 min in TBS.Primary rat hippocampal neuron cultures were prepared as described above. Forty thousand cells were plated into each well of an eight-well chamber slide (Ibidi) coated with poly-D-lysine, and the cultures were treated with lentivirus at 0.25 pg of p24 protein/plated cell on day 2. The number of puncta/total area of Tau12 signal (nm2) was calculated for each image with a minimum cutoff of five pixels. The individual data points (see C) represent the mean of the results from all analyzed images within an individual replicate, and the experiment was repeated three independent times.The cells were imaged on a Nikon A1+ laser scanning confocal microscope equipped with 488, 561, and 640 solid state lasers using a 60× 1.40 numerical aperture (NA) objective and Nikon Elements AR software. We collected z-stack images over a depth of 2 µm with 0.25 µm steps (nine images/stack). The displayed images represent maximum intensity projections from the individual z-stack planes. Using a 40× 1.3 NA objective, we collected two to six images containing randomly selected, isolated neurons expressing exogenous tau (Tau12-positive) for each condition and replicate. Monochromatic images were produced for each channel, and the data were quantified using FIJI ImageJ. For the PLA puncta image (green channel) a threshold mask was generated using the default threshold setting, and the number of puncta per image was counted using the Count Particle function. The human tau signal (red channel) was identified using post-PLA immunocytofluorescence staining using the Tau12 antibody to label only exogenous tau as described above. A thresholding mask was generated using the Huang setting, and the total area of the mask was calculated using the Measure function and converted from pixels to nmints see C represem NaCl for each well; 0.5 µg of DNA was also added to 50 µl of 150 mm NaCl. The PEI mix was added to the DNA mix, vortexed briefly, and incubated at room temperate for 20 min. The samples were then added to the wells in a dropwise manner. The lysates were collected in 75 µl of lysis buffer/well , and two wells for each condition were combined and sonicated with four pulses. Lysate protein concentrations were determined using the Bio-Rad protein assay as directed. Immunoblotting was performed as described above by loading 40 μg of total protein into 18-well gels that were transferred to nitrocellulose.Low-passage HEK293T cells were plated into a poly-D-lysine-coated 24-well plate at 200,000 cells/well . After 24 h the cells were transfected with 0.5 µg of DNA. Polyethylenimine (PEI) was incubated at 55°C for 5 min before 5 µl was added to 50 µl of 150 mPrimary rat hippocampal neurons (E18) were plated at 600,000 cells/well into six-well plates coated with poly-D-lysine as described above. On DIV 4 the neurons were treated with lentivirus (0.5 pg p24/cell) to induce expression of WT tau, P301L tau, R5L tau, or GFP. On DIV 8 lysates from individual wells were collected into 200 μl of the same lysis buffer described above and sonicated with four pulses. Cell lysate  was loaded onto a 12-well gel for the PP1 blots and an 18-well gel  for the tau and GFP blots, then transferred to nitrocellulose. The primary neuron blots were blocked in 2% milk-TBST for 1 h, washed 3× in TBST, then incubated with primary antibodies in 5% BSA-TBST overnight at 4°C.AB_330823), HaloTag , Total PP1 , GAPDH , Tau5 . Blots were detected with secondary antibodies and imaged as described above. The pT320 PP1 (inactive form) signal was normalized to total PP1  to obtain relative changes in the levels of active PP1. Notably, T320 in PP1α is analogous to T316 in PP1β and T311 in PP1γ. Tau signal was normalized to GAPDH signal to confirm similar levels of protein expression and loading.Blots were probed for phospho-PP1  as described previously (AB_2633277).Primary hippocampal neurons were prepared as described earlier and plated at 80,000 cells/well into four-well live cell chamber slides (Ibidi) coated with poly-D-lysine in 750 μl of neurobasal media (Invitrogen). The cells were transfected on DIV 8 using Lipofectamine 2000 reagent. A pFIN Synaptophysin-mApple and a pCMV plasmid containing one of the genes of interest  were coincubated at 200 ng each in 30 µl of Opti-MEM serum-free media for 30 min. For each well, 0.5 µl of Lipofectamine2000 was also incubated in 30 µl of Opti-MEM media (Invitrogen) at room temperature for 30 min. The Lipofectamine mix was added to the DNA mix and incubated for another 30 min at room temperature. Sixty microliters of sample was added throughout the entirety of each well. After 2 h, half of the media (380 µl) was replaced with an equivalent volume of neurobasal media containing an antibody that binds the extracellular domain of neurofascin in the axon initial segment to differentiate the axon from dendrites during live cell imaging  for 5 min using the 568 nm laser to visualize movement of mApple-synaptophysin cargo. Kymographs were generated using the ImageJ macro KymoAnalyzer ; 400 ng of a pH1SH plasmid including shRNA sequences for PP1α, PP1γ, Renilla luciferase (positive control); a nontargeting control sequence (negative control); or empty for normalization. Two days after transfection, the cell culture media was replaced by 200 μl of Passive Lysis Buffer, and the cells were incubated on a shaker for 15 min. Then 20 μl of lysate was transferred to white opaque 96-well plates in triplicate and placed in a Promega GloMax-Multi Detection System. Following the Dual Luciferase two-injector protocol, the instrument injected 100 μl of Luciferase Assay Reagent II and measured the firefly luciferase activity. It then dispensed 100 μl of Stop & Glo Reagent and measured the Renilla luciferase activity. The Renilla:Firefly luciferase activity signal ratios were normalized to the average of the relevant Renilla fusion construct + empty pH1SH vector and reported as percent of control. This indicates the percentage knockdown of PP1 expression for each of the PP1 and control shRNA constructs.Knockdown of PP1 was evaluated using a Dual-Luciferase Reporter Assay System (Promega). HEK293T cells were plated into a poly-D-lysine-coated 24-well plate at 150,000 cells/well and transfected the next day. The transfection included 100 ng of a fusion construct containing m DTT, 1× concentration of rat PP1α or PP1γ isoform-specific PCR primers and HEX fluorescent probe, and a 1× concentration of rat rplp0 PCR primers and FAM fluorescent probe as a reference gene. Oil droplets were generated using a QX200 automatic droplet generator (Bio-Rad), and 40 μl of droplets were transferred to a ddPCR 96-well plate (Bio-Rad), and the plate was covered with a foil cover and sealed. The reverse transcriptase reactions occurred at 50°C for 1 h in a C1000 Bio-Rad thermocycler. The PCR amplification protocol was 95°C for 10 min to activate the enzyme followed by 95°C for 30 s and 60°C for 60 s, repeated 40× with ramp rates of 3°C/s. The enzyme was deactivated at 98°C for 10 min and held at 4°C. A QX200 Droplet Reader (Bio-Rad) was used to quantify droplet fluorescence for each probe using the Absolute quantitation function of QuantaSoft software (Bio-Rad). The software calculated concentrations of PP1 and rplp0 RNA (copies/μl) and reported as a ratio of PP1 to the reference gene.We also confirmed mRNA knockdown in primary neurons using droplet digital PCR (ddPCR). Primary rat hippocampal neurons were plated at 150,000 cells/well into a 24-well plate and individually transduced on DIV four with lentiviruses to express shRNA targeting PP1α, PP1γ, or a scrambled shRNA control. RNA was extracted using a Qiagen RNeasy miniprep kit 8 d after transduction. The relative concentrations of PP1α and PP1γ mRNA were quantified using ddPCR. One nanogram of extracted RNA from each sample was diluted into a reaction mix as directed using the One-Step RT-ddPCR Advanced Kit for Probes (Bio-Rad). The reaction mix contained a final 1× Supermix concentration, 20 U/μl of reverse transcriptase, 15 mn = 6, 11 neurons; WT tau + PP1α shRNA, n = 6, 9 neurons; WT tau + PP1γ shRNA, n = 6, 10 neurons; P301L tau + Control shRNA, n = 6, 12 neurons; P301L tau + PP1α shRNA, n = 6, 10 neurons; P301L tau + PP1γ shRNA, n = 6, 9 neurons; R5L tau + Control shRNA, n = 6, 10 neurons; R5L tau + PP1α shRNA, n = 6, 10 neurons; R5L tau + PP1γ shRNA, n = 6, 9 neurons.The neurons were transfected with 175 ng of pCMV tau or GFP DNA, 175 ng of pH1SH shRNA DNA, and 100 ng of mApple-Synaptophysin DNA using Lipofectamine 2000, and the transport data collection and analysis was conducted using conditions identical to those described above. The number of independent experimental replicates and total number of neurons analyzed were as follows: WT tau + Control shRNA, post hoc multiple comparison test , as previously reported (m monomeric P301L tau (B) or R5L tau (C). We calculated the means of the rate readings over the last 20 min of the assay to identify any differences among the conditions. Perfusion with P301L or R5L mutant tau significantly impaired the rate of anterograde FAT when compared with WT tau  = 25.25, p = 0.0001; P301L vs WT, p = 0.0082; R5L vs WT, p = 0.0001). P301L and R5L tau perfusion also reduced retrograde FAT rates compared with WT tau  = 16.68, p = 0.0007; P301L vs WT, p = 0.0079; R5L vs WT, p = 0.0008). These data demonstrate that in the isolated axoplasm preparation, both the P301L and R5L mutations confer on monomeric tau a toxic effect on FAT.Using vesicle motility assays in isolated squid axoplasm, we first tested whether monomeric P301L or R5L mutant tau affects FAT. Following perfusion of 2 μntration , FAT ratntration A, as prereported . In cont301L tau B or R5L  R5L tau C. We calA). Immunoblotting experiments showed no significant differences in tau or PP1 expression across the conditions . All tau proteins eluted in the Halo-PP1α pull-down elution sample , but not with HaloTag alone . Quantitative data showed increased P301L tau levels in pull-down elution, compared with WT and R5L tau, indicating enhanced binding of mutant P301L tau to PP1α  = 7.348, p = 0.0244; PP1α, F = 11.46, p = 0.0429; Interaction, F = 7.339, p = 0.0244; P301L vs WT tau, p = 0.0311; P301L vs R5L tau, p = 0.0198). We normalized the tau signal to the change in Halo signal from before to after pull down  because the HaloTag covalently binds the resin and does not elute in these conditions. In the Halo-PP1β pull-down samples, we detected trace amounts of tau , but the degree of interaction was about fivefold lower than with PP1α or PP1γ. The P301L tau mutation did not significantly increase the interaction with PP1β compared with WT tau  = 5.093, p = 0.0509; PP1β, F = 11.51, p = 0.0427; Interaction, F = 5.765, p = 0.0401; P301L vs WT tau, p = 0.1020) but did compared with R5L tau (p = 0.0287). Finally, all three tau forms eluted with the pull downs of the Halo-PP1γ isoform . Again, P301L tau induced a significant increase in binding efficiency compared with WT and R5L tau  = 17.28, p = 0.0032; PP1γ, F = 30.89, p = 0.0115; Interaction, F = 16.86, p = 0.0034; P301L vs WT tau, p = 0.0033; P301L vs R5L tau, p = 0.0008). Notably, the level of R5L pulled down with PP1α (p = 0.9962), PP1β (p = 0.8256), or PP1γ (p = 0.9999) was unchanged when compared with WT, suggesting that this mutation does not alter the extent of physical interaction between tau and PP1. We did not detect tau in any of the control samples in concurrent pull downs that were performed with HaloTag-only , which effectively controls for nonspecific binding of HaloTag protein, nonspecific interactions with the Halo-Link resin, and other experimental conditions. Collectively, these data suggest that tau interacts with PP1α and PP1γ and that this interaction is significantly enhanced by the pathogenic P301L tau mutation but not the R5L mutation.Next, we sought to evaluate whether tau interacts with any of the three major PP1 isoforms in mammalian nerve tissues, PP1α, PP1β, and PP1γ1 . We begaFig. 2A). This complementary approach avoids issues related to cell lysis before assessing the interaction. First, we performed a donor saturation assay by transfecting the cells with a constant concentration of transfected donor DNA and varied concentrations of acceptor DNA. A specific interaction is indicated by high-signal, hyperbolic curves that display saturation of the nBRET ratios when plotted against the DNA ratio (B). In contrast, nBRET ratios generated from cells expressing Halo-PP1β with any of the tau constructs did not support interaction between the proteins because of the overall low nBRET signal (C). Coexpression of Halo-PP1γ with tau produced curves indicating a specific interaction with each of the tau forms (D). The low level of interaction with PP1β compared with PP1α and PP1γ aligns well with data from co-pull-down experiments (E).Results from pull-down experiments provide evidence supporting a protein–protein interaction between tau and selected PP1 isoforms. We used nBRET assays to further evaluate this interaction in living cells A. This cNA ratio . The nBRNA ratio B. In conT signal C. Coexprau forms D. The loeriments . To conteriments E.F; Tau, F = 58.78, p < 0.0001; Halo-PP1α, F = 273.8, p < 0.0001; Interaction, F = 47.98, p < 0.0001; P301L vs WT tau, p < 0.0001; P301L vs R5L tau, p < 0.0001; Halo-PP1α vs Halo-only WT tau, p = 0.0006; P301L tau, p < 0.0001; R5L tau, p = 0.0006). None of the individual comparisons with Halo-PP1β were statistically significant, again suggesting a weak or nonexistent interaction with this PP1 isoform  = 2.381, p = 0.1209; Halo-PP1β, F = 6.149, p = 0.0233; Interaction, F = 0.8514, p = 0.4433). Similar to PP1α, the nBRET ratio of P301L tau and Halo-PP1γ significantly increased when compared with WT and R5L tau, and all three tau proteins significantly increased the ratio with Halo-PP1γ coexpression compared with Halo-only controls  = 60.49, p < 0.0001; Halo-PP1γ, F = 225.7, p < 0.0001; Interaction, F = 50.36, p < 0.0001; P301L vs WT tau, p < 0.0001; P301L vs R5L tau, p < 0.0001; Halo-PP1γ vs Halo-only WT tau, p = 0.0009; P301L tau, p < 0.0001; R5L tau, p = 0.0079). The nBRET ratios for R5L were similar to those for WT with PP1α (p = 0.9610), PP1β (p = 0.9998) or PP1γ (p = 0.9739), confirming that this mutation does not alter the degree of interaction in an in-cell assay. Considered together, these data indicate that when coexpressed in living cells, tau interacts with PP1α and PP1γ but only very weakly or not at all with PP1β. Additionally, the P301L mutation robustly enhances the interaction with PP1α and PP1γ compared with WT tau, whereas the R5L mutation does not, which is consistent with results from experiments in We repeated the nBRET assay in four independent experiments at a single DNA ratio of 100:1 to perform statistical comparisons among tau groups and among Halo-PP1 and the Halo-only control. The nBRET ratios with P301L tau and Halo-PP1α significantly increased compared with WT and R5L tau with Halo-PP1α and significantly increased for all tau groups compared with the Halo-only control groups . Following amplification and hybridization with fluorescent oligonucleotides, ligated oligos are indicated by bright puncta .After using an immortalized cell line to identify mutation- and isoform-specific effects, we next wanted to identify interactions in neurons. Using lentiviruses, we expressed the various tau constructs in primary rat hippocampal neuron cultures, which typically produce low levels of overexpression. We used PLA to identify relative differences in the association between tau and PP1 in the neurons expressing WT, P301L, and R5L tau. The assay uses antibody pairs that selectively recognize putative interactor proteins. Secondary antibodies, linked to complementary oligonucleotide sequences, bind the primary antibodies. The complementary oligo sequences are ligated t puncta A, green.B). Quantification of the PLA signal indicated that the P301L mutation enhanced the association between tau and PP1 compared with WT and R5L tau  = 42.97, p = 0.0020; P301L vs WT tau, p = 0.0021; P301L vs R5L, p = 0.0046). Neurons expressing WT human tau presented PLA puncta, indicating a tau-PP1 association in neurites and cell bodies . We identified increased concentrations of puncta in neurons expressing P301L tau that also localized to neurites and soma . R5L-expressing neurons also displayed puncta with similar patterns of subcellular localization, but there was no increase compared with WT . The control conditions included primary antibody deletions of Tau7 , PP1 , and both antibodies , all of which showed little to no background signal in the neurons. Finally, we identified associations between endogenous rat tau and PP1 in nontransduced cells . These results strongly support an interaction between tau and PP1 in mammalian neurons under physiological conditions. WT tau and both mutants interact with PP1, but the P301L mutation increases the binding of PP1 to tau relative to WT, whereas the R5L mutation does not increase PP1 binding over WT tau.We probed neurons with Tau7, a mouse monoclonal antibody that binds to the C terminus of tau  and a PPFig. 4A,B; F = 2.764, p = 0.1610). Next, we probed lysates by immunoblotting using an anti-pT320 PP1 antibody that recognizes the inhibitory phosphoT320 site in PP1α and analogous sites T316 in PP1β and T311 in PP1γ  = 4.705, p = 0.0885; P301L vs WT tau, p = 0.0031) but none of the other comparisons were statistically different . We confirmed similar levels of tau expression in the PP1β experiments  = 0.9685, p = 0.4025). Expressing each of the mutant tau proteins led to a significant increase in the level of active PP1β when compared with GFP and WT tau  = 41.45, p = 0.0008; P301L tau vs GFP, p = 0.0216; R5L tau vs GFP, p = 0.0029; P301L vs WT tau, p = 0.0236; R5L vs WT tau, 0.0211). We then expressed similar levels of tau with PP1γ  = 1.128, p = 0.3696). Again, both P301L and R5L mutant tau proteins significantly increased the levels of active PP1γ compared with GFP and WT tau  = 30.28, p = 0.0103; P301L tau vs GFP, p = 0.0262; R5L tau vs GFP, p = 0.0328; P301L vs WT tau, p = 0.0070; R5L vs WT tau, p < 0.0001). These results show that P301L or R5L mutant coexpression with PP1 reduced levels of T320 phosphorylation in PP1β and PP1γ isoforms compared with control conditions , and P301L tau expression reduced it in PP1α compared with GFP expression. This suggests that these FTLD-tau mutants are associated with increased levels of active PP1 in HEK293T cells.After establishing tau interactions with PP1α and PP1γ, we determined whether tau also affected levels of active PP1 in cells cotransfected with GFP, WT, P301L, or R5L tau. First, we confirmed that similar levels of tau expression, normalized to GAPDH levels, were present in the experiments with PP1α coexpression  = 3.013, p = 0.1632). Then we probed the lysates with the anti-pT320 PP1 antibody and a total PP1 antibody . Expression of P301L tau and R5L tau increased levels of active PP1 (indicated by decreased inactive pT320 PP1) compared with GFP-expressing neurons  = 7.053, p = 0.0420; P301L tau vs GFP, p = 0.0403; R5L tau vs GFP, p = 0.0468). In contrast, WT tau expression did not significantly alter active PP1 levels (p = 0.9962). This supports an increase in active PP1 levels following expression of FTLD mutant tau, but not WT tau.After identifying the effect of tau on active PP1 levels in the HEK293T cell line, we set out to measure these effects on endogenous PP1 in primary rat hippocampal neurons by individually expressing the three tau constructs or a GFP control via lentiviral transduction. We immunoblotted the neuron lysates to confirm equal levels of protein loading and tau expression . Using live cell confocal microscopy to image vesicle transport along the axon  we then measured transport characteristics via kymograph analysis . In GFP-expressing and all tau-expressing neurons, fluorescently tagged synaptic vesicle precursors displayed similar anterograde FAT rates  = 1.369, p = 0.2824). When compared with GFP cells, we detected a small but significant increase in retrograde FAT rates (∼20%) in neurons expressing each of the tau proteins  = 5.900, p = 0.0051; WT tau vs GFP, 0.0353; P301L tau vs GFP, p = 0.0103; R5L tau vs GFP, 0.0104). We also analyzed the impact of tau expression on the pause frequency of fluorescently labeled vesicles, as PP1 activation by pathogenic tau promotes detachment of kinesin-1 from its transported organelle cargoes  = 32.58, p < 0.0001; WT tau vs GFP: p = 0.9034). In contrast, P301L tau expression significantly increased the frequency of cargo pauses compared with both GFP (p < 0.0001) and WT tau . To evaluate directionality-related effects on FAT, we further separated total pause frequency data for cargo traveling in the anterograde or retrograde directions. Expression of P301L tau or R5L tau significantly increased anterograde pause frequency compared with both GFP- and WT tau-expressing neurons  = 5.990, p = 0.0047; P301L vs GFP, 0.0471; P301L vs WT tau, p = 0.0345; R5L tau vs GFP, p = 0.0341; R5L tau vs WT tau, p = 0.0252). Retrograde pause frequency also increased with expression of P301L tau compared with GFP (p = 0.0064) and WT tau  = 8.853, p = 0.0007) and R5L tau induced the same effects . These data suggest that although WT tau expression does not have an impact on cargo pausing, mutant P301L tau and R5L tau expression significantly increase cargo pause frequency. On the other hand, FAT rates were unaltered (anterograde FAT) or slightly increased (retrograde FAT) by all three forms of tau compared with control conditions.Next, we tested whether our findings about the effects of tau on FAT in the squid axoplasm extended to mammalian cultured neurons. We cotransfected rat primary hippocampal neurons with mApple-synaptophysin (an established cargo protein of synaptic vesicle precursors) and either WT, P301L, or R5L mutant tau or a GFP control construct A. Using  cargoes . CompareRenilla luciferase-PP1 isoform fusion construct in a dual luciferase assay. The PP1α shRNA produced 87% knockdown when compared with controls expressing the reporter. The control nontargeting shRNA did not significantly alter expression when compared with the empty controls . The PP1γ shRNA produced 90% knockdown, whereas the signal with control shRNA was 102% of empty vector controls . To confirm knockdown in rat primary neurons we transduced them with lentiviruses expressing PP1α-shRNA, PP1γ-shRNA, or control shRNA. Using ddPCR we determined the mRNA concentrations from cell lysates under each condition, then normalized them to rplp0 reference gene concentrations. The ddPCR analysis showed that PP1α mRNA levels were reduced 71% by PP1α-specific shRNA and only 19% by PP1γ-specific shRNA compared with the control shRNA . Similarly, PP1γ mRNA levels were reduced 70% by PP1γ-specific shRNA expression and only 9% by PP1α-specific shRNA when compared with the control shRNA treatment . These data validate that each PP1 isoform-specific shRNA produces target knockdown with low off-target  effects in cultured hippocampal neurons.Next, we determined whether the P301L tau-induced effect on FAT documented in E; Tau, F = 1.128, p = 0.3325; shRNA, F = 5.053, p = 0.0105; Interaction, F = 0.1333, p = 0.9693). R5L tau produced a small but significant increase in retrograde FAT compared with WT tau in the control shRNA condition  = 1.999, p = 0.1473; shRNA, F = 3.788, p = 0.0302; Interaction, F = 1.148, p = 0.3464; R5L vs WT tau, p = 0.0333). These data indicate that exogenous tau expression did not affect FAT rates significantly, regardless of shRNA treatment groups.Having established the knockdown specificity and effectiveness of shRNAs, we performed triple transfections of primary rat hippocampal neurons aimed at coexpressing (1) shRNA targeting PP1α or PP1γ or the nontargeting control shRNA; (2) WT, P301L, or R5L tau; and (3) mApple-synaptophysin in the various combinations. The PP1 isoform-specific shRNAs did not significantly affect anterograde transport velocity  = 17.61, p < 0.0001; shRNA, F = 16.61, p < 0.0001; Interaction, F = 4.463, p = 0.0040). Compared with expression of WT tau, the PP1α shRNA did not significantly modify the increase in total pause frequency induced by mutant tau expression . In contrast, PP1γ shRNA significantly reduced the total pause frequency in P301L- and R5L-expressing neurons to levels observed in WT neurons with control shRNA . These data indicate that the increase in FAT pause frequency induced by P301L or R5L tau expression is eliminated by specific knockdown of PP1γ, but not PP1α.We next examined whether PP1 shRNAs abolished the mutant-tau-induced effects on cargo pausing frequency. As expected from results in Fig. 7G–I), expression of P301L tau significantly increased anterograde pause frequency  = 15.80, p < 0.0001; shRNA, F = 20.94, p < 0.0001; Interaction, F = 2.133, p = 0.0923) compared with cells expressing WT tau with control shRNA treatment. Knockdown of PP1α did not alter this effect. In contrast, treating the P301L- or R5L-expressing neurons with PP1γ-shRNA eliminated the increased anterograde pause frequency, restoring pausing to WT tau levels .To further investigate the specificity of mutant tau effects on cargo pausing and the relationship of these effects to PP1γ knockdown, pause frequencies were binned for cargo traveling in the anterograde or retrograde FAT directions. Here, we found that PP1γ knockdown rescued mutant-tau-induced pausing defects in the anterograde direction. As observed with no shRNA treatments G–I, exprI; F = 5.193, p = 0.0093; shRNA, F = 9.244, p = 0.0004; Interaction, F = 3.921, p = 0.0082). As expected, P301L or R5L expression increased retrograde cargo pause frequency with control shRNA coexpression. We did not observe significant differences among any of the tau groups with knockdown of PP1α, likely because of the nonsignificant elevation in pause frequency noted with PP1α shRNA . Again, PP1γ shRNA completely prevented the increase in retrograde pause frequency in P301L- and R5L-expressing neurons. Collectively, these data indicate that P301L and R5L tau produce pause frequency abnormalities in both directions and that specifically knocking down PP1γ robustly alleviates these abnormalities.We detected similar effects of tau expression on retrograde pause frequency, albeit lower in magnitude . Expression of P301L tau in the neurons increased PLA puncta compared with WT tau expression  = 8.785, p = 0.0344; P301L vs WT tau, p = 0.0326). No significant differences existed between WT tau and R5L tau (p = 0.5210). Primary deletion of the various antibodies resulted in little to no background signal . WT tau-expressing neurons displayed PLA puncta , present in the neuronal processes  and cell bodies  indicating an association with the PP1γ isoform specifically. Expression of mutant P301L tau increased the number of puncta compared with WT tau expression . R5L tau also interacted with PP1γ in the neurons at similar levels to WT tau . Notably, these findings mirror the neuron PLA data using a pan-PP1 antibody . Together, this supports a neuronal association between tau and the PP1γ isoform, found to be the mediator of the mutant-tau-induced effects on FAT , PP1β , and PP1γ . Expressing Δ2–18 WT tau and Δ2–18 P301L tau resulted in reduced active PP1α  compared with full-length counterparts . Importantly, PAD deletion rescued the P301L-induced change from WT tau . Similarly, P301L tau resulted in an increase in active PP1β compared with WT tau (p = 0.0232), and deletion of PAD reduced active PP1β for both WT and P301L tau . Finally, we detected lower levels of active PP1γ with Δ2–18 WT tau compared with full-length tau expression . P301L tau induced a significant increase in active PP1 compared with WT tau (p = 0.0092), and PAD deletion in a P301L background caused a ninefold reduction in active PP1γ that did not reach statistical significance (p = 0.1531) and a return to levels that were not significantly different from WT tau (p = 0.7579). These indicate that generally the PAD region in the N terminus of tau mediates its effects on increasing levels of active PP1.Previously, the PAD (amino acids 2–18 in tau) was identified as necessary and sufficient to induce PP1-dependent transport defects in the squid axoplasm . DiseaseFig. 9A; F = 2.431, p = 0.1029) or retrograde  = 0.2275, p = 0.8758) velocities. Again, P301L tau increased total pause frequency compared with WT tau  = 13.59, p = 0.0001; WT tau vs P301L tau, p = 0.0009) and PAD deletion rescued pause frequency to control levels . We observed a similar effect on pause frequency in the anterograde direction whereby PAD deletion rescued the P301L-induced increases  = 7.150, p = 0.0029; WT tau vs P301L tau, p = 0.0281; WT tau vs Δ2–18 P301L tau, p = 0.8536; P301L tau vs Δ2–18 P301L tau, p = 0.0056). We did not detect significant increases in retrograde pause frequency in this experiment  = 3.080, p = 0.0574; WT tau vs P301L tau, p = 0.1963) but deletion of amino acids 2–18 in P301L tau significantly reduced pause frequency compared with full-length P301L tau expression (p = 0.0479). This indicates that alterations in FAT elicited by P301L are PAD dependent, as previously shown for other pathogenic forms of tau in the isolated squid axoplasm model  alter PP1γ function resulting in dysregulation of FAT . Given t


N-acylethanolamine acid amidase (NAAA) is an N-terminal nucleophile (Ntn) hydrolase that catalyses the intracellular deactivation of the endogenous analgesic and anti-inflammatory agent palmitoylethanolamide (PEA). NAAA inhibitors counteract this process and exert marked therapeutic effects in animal models of pain, inflammation and neurodegeneration. While it is known that NAAA preferentially hydrolyses saturated fatty acid ethanolamides (FAEs), a detailed profile of the relationship between catalytic efficiency and fatty acid-chain length is still lacking. In this report, we combined enzymatic and molecular modelling approaches to determine the effects of acyl chain and polar head modifications on substrate recognition and hydrolysis by NAAA. The results show that, in both saturated and monounsaturated FAEs, the catalytic efficiency is strictly dependent upon fatty acyl chain length, whereas there is a wider tolerance for modifications of the polar heads. This relationship reflects the relative stability of enzyme-substrate complexes in molecular dynamics simulations. N-acylethanolamine acid amidase (NAAA) is a cysteine hydrolase that catalyses the intracellular degradation of the lipid-derived messenger, palmitoylethanolamide (PEA), to palmitic acid and ethanolamine,,N-terminal nucleophile (Ntn) superfamily of enzymesN-terminal amino group of the catalytic cysteine, with the carbonyl oxygen stabilised by the interaction with the oxyanion hole residues.,N-acylphosphatidylethanolamine phospholipase D, NAPE-PLD) that cleaves the glycerophospholipid precursor, N-palmitoylphosphatidylethanolamine, to produce FAE and phosphatidic acid,NAAA’s best known substrate, PEA,N-acyltaurinesN-acylglycinesIn addition to NAAA, the hydrolysis of PEA is catalysed by fatty acid amide hydrolase (FAAH)N-acylethanolamines. More specifically, the relative reactivities of the enzyme were found to be at 100% for PEA, 48% for N-myristoylethanolamine, approximately 20% for N-stearoylethanolamine and N-oleoylethanolamine, 13% for N-linoleoylethanolamine and only 8% for anandamide,In the present study, we used a combination of enzyme kinetic and computational analyses to conduct a systematic investigation of the substrate selectivity of NAAA. The work of Ueda and collaborators has provided evidence for a marked selectivity of NAAA for PEA compared to other Oryctolagus cuniculus, rabbit NAAA) in complex with a molecule of myristic acid was selected as template structure for homology modelling of rNAAA (rat NAAA). The X-ray structure of ocNAAA and the amino acid sequence of ocNAAA and rNAAA were retrieved from the Protein Data Bank (PDB 6DY1),The ocNAAA , prepared as follows: 3.7 ml of p-anisaldehyde were added to 135 ml of absolute ethanol containing 5 ml of concentrated sulphuric acid and 1.5 ml of glacial acetic acid. The staining solution was stored in a jar covered with aluminium foil at 4 °C. 1H NMR spectra were recorded on a Bruker DRX400 (400 MHz). Chemical shifts  are reported in parts per million (ppm), referenced to tetramethylsilane  or the residual solvent signal . 1H NMR spectra are reported in the following order: number of protons, multiplicity and approximate coupling constant  in hertz (Hz), and a clear attribution of proton signals is provided. Signal multiplicities were characterised as s (singlet), d (doublet), dd (doublet of doublets), t (triplet), dt (doublet of triplets), q (quartet), quint (quintet) m (multiplet), br . 1H NMR spectra are collected in Supporting Information. Mass spectra were recorded on an Agilent 6410 Triple Quad LC/MS system with an ESI interface. The purity of final compounds was assessed by liquid chromatography/tandem mass spectrometry (LC/MS-MS) using an Agilent 6410 Triple Quad system (Agilent Technologies). Prior to analyses, samples were prepared in methanol at a final concentration of 5 µg mL−1. Analytes separation was performed on an Agilent Zorbax Eclipse XBD C18 column  by gradient elution. Flow rate was set at 0.4 ml min−1, and the injection volume was 2.0 µL. Solvent A was water and solvent B was methanol, both added of 0.25% v/v acetic acid and 5 mM ammonium acetate. Gradient conditions A: t (0 min): 10% A: 90% B; t (2 min): 5% A: 95% B; t (3 min): 10% A: 90% B. All tested compounds were >95% pure. Specific rotation  analysis was conducted on HPTLC aluminium sheets ; compounds were visualised by dipping in a solution of 1–3 and 5–6 : to an ice-cooled stirring solution of the fatty acid (0.725 mmol) and triethylamine (1.813 mmol) in dichloromethane (11 ml), 1-hydroxybenzotriazole  and N,N,N’,N’-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate  were added and the reaction was stirred for 60 min. Ethanolamine (0.870 mmol) was added. After 2 h the reaction was quenched with hydrochloric acid (1 M), and the organic phase was washed with a saturated solution of sodium bicarbonate, then water, and brine. The organic layer was treated with magnesium sulphate then filtered and dried under vacuum. The crude product was recrystallised from heptane or purified by column chromatography (isocratic elution with a mixture of chloroform:methanol (98:2 v/v)) to give the desired compound.Compounds +. 1H-NMR : δ 6.17 , 3.71 , 3.40 , 3.22 , 2.20 , 1.63 , 1.38–1.14 , 0.88 .Procedure A, tridecanoyl chloride (0.37 g), ethanolamine (0.19 µL), yield = 405 mg (> 98%) . MS calc.: 257.24, found: 258.3 m/z [M + H]+. 1H-NMR : δ 6.03 , 3.72 , 3.41 , 2.91 , 2.20 , 1.64 , 1.39–1.13 , 0.88 . Analytical data are consistent with those previously reported in the literature,Procedure A, tetradecanoyl chloride (0.39 g), ethanolamine (0.21 µL), yield = 420 mg (98%) . MS calc.: 271.25, found: 272.3 m/z [M + H]+. 1H-NMR : δ 6.05 , 3.72 , 3.42 , 2.96 , 2.20 , 1.63 , 1.40–1.13 , 0.88 . Analytical data are consistent with those previously reported in the literatureProcedure A, pentadecanoyl chloride (0.42 g), ethanolamine (0.20 µL), yield = 437 mg (95%) . MS calc.: 285.27, found: 286.3 m/z [M + H]+. 1H-NMR : δ 5.97 , 3.72 , 3.42 , 2.79 , 2.20 , 1.63 , 1.40–1.15 , 0.88 . Analytical data are consistent with those previously reported in the literatureProcedure B, palmitic acid (0.19 g), TBTU (0.26 g), HOBt (0.11 g), Et3N (250 µL), ethanolamine (53 µL), purified by recrystallisation from heptane, yield = 139 mg 64%. MS calc.: 299.28, found: 300.3 m/z [M + H]+. 1H-NMR : δ 5.96 , 3.72 , 3.42 , 2.75 , 2.20 , 1.63 , 1.40–1.11 , 0.88 . Analytical data are consistent with those previously reported in the literatureProcedure A, heptadecanoyl chloride (0.48 g), ethanolamine (0.20 µL), yield = 453 mg (87%) . MS calc.: 313.30, found: 314.3 m/z [M + H]+. 1H-NMR : δ 5.89 , 3.80 − 3.65 , 3.43 , 2.60 , 2.20 , 1.63 , 1.40–1.12 , 0.88 . Analytical data are consistent with those previously reported in the literatureProcedure A, octadecanoyl chloride (0.49 g), ethanolamine (0.21 µL), yield = 519 mg (98%) . MS calc.: 327.31, found: 328.3 m/z [M + H]Z)-pentadec-10-enoic acid (0.17 g), TBTU (0.27 g), HOBt (0.11 g), Et3N (252 µL), ethanolamine (52 µL), purified by column chromatography, yield = 108 mg (53%). MS calc.: 283.25, found: 284.3 m/z [M + H]+. 1H-NMR : δ 5.93 , 5.40–5.28 , 3.72 , 3.42 , 2.67 , 2.20 , 2.08–1.88 , 1.63 , 1.40–1.19 , 0.89 .Procedure B, (Z)-hexadec-9-enoic acid (0.19 g), TBTU (0.27 g), HOBt (0.12 g), Et3N (263 µL), ethanolamine (54 µL), purified by column chromatography, yield = 110 mg (49%). MS calc.: 297.27, found: 298.3 m/z [M + H]+. 1H-NMR : δ 6.01 , 5.40–5.28 , 3.72 , 3.42 , 2.89 , 2.20 , 2.06–1.93 , 1.63 , 1.39–1.17 , 0.88 .Procedure B, (Z)-heptadec-10-enoic acid (0.18 g), TBTU (0.23 g), HOBt (0.10 g), Et3N (230 µL), ethanolamine (48 µL), purified by column chromatography, yield = 120 mg (58%). MS calc.: 311.28, found: 312.3 m/z [M + H]+. 1H-NMR : δ 6.01, 5.40–5.28 , 3.72 , 3.41 , 2.89 , 2.20 , 2.08–1.91 , 1.63 , 1.40–1.15 , 0.88 .Procedure B, (Z)-nonadec-10-enoic acid (0.19 g), TBTU (0.23 g), HOBt (0.10 g), Et3N (228 µL), ethanolamine (47 µL), purified by column chromatography, yield = 129 mg (61%). MS calc.: 325.30, found: 326.3 m/z [M + H]+. 1H-NMR : δ 6.03 , 5.42–5.26 , 3.72 , 3.41 , 2.85 , 2.20 , 2.09–1.90 , 1.63 , 1.48–1.06 ppm , 0.88 ppm . Analytical data are consistent with those previously reported in the literatureProcedure B, (Z)-nonadec-10-enoic acid (0.21 g), TBTU (0.25 g), HOBt (0.11 g), Et3N (251 µL), ethanolamine (52 µL), purified by column chromatography, yield = 80 mg (33%). MS calc.: 339.31, found: 340.3 m/z [M + H]+. 1H-NMR : δ 5.89 , 5.40–5.28 , 3.72 ppm , 3.42 ppm , 2.60 , 2.20 ppm , 2.08–1.91 , 1.63 ppm , 1.40–1.20 ppm , 0.88 ppm .Procedure B, (Z)-eicos-9-enoic acid (0.23 g), TBTU (0.26 g), HOBt (0.11 g), Et3N (260 µL), ethanolamine (54 µL), purified by column chromatography, yield = 107 mg (41%). MS calc.: 353.33, found: 354.3 m/z [M + H]+. 1H-NMR : δ 5.98 ppm , 5.39–5.24 , 3.72 ppm , 3.42 ppm , 2.81 , 2.20 ppm , 2.06–1.91 , 1.63 ppm , 1.38–1.18 ppm , 0.88 ppm .Procedure B, . MS calc.: 255.26, found: 256.3 m/z [M + H]+. 1H-NMR : δ 5.35 ppm , 2.22 ppm , 1.64 ppm , 1.40–1.14 ppm , 0.88 ppm . Analytical data are consistent with those previously reported in the literatureTo a solution of palmitic acid  in ice-cold dichloromethane (12 ml), triethylamine , HOBt  and TBTU  were added sequentially, and the mixture was stirred for 30 min. Then ammonium chloride  was added to the reaction, that was stirred overnight (synthetic schemes are collected in the Supporting Information). The mixture was then diluted with dichloromethane and washed with HCl 1 M, then saturated NaHCO3, and brined. The organic phase was dried over MgSO4, and the crude product was recrystallised from heptane to get 139 mg of target product (yield = 63%). MS calc.: 269.27, found: 270.3 m/z [M + H]+. 1H-NMR : δ 5.39 ppm , 2.38 , 2.16 ppm , 1.62 ppm , 1.38–1.15 ppm , 0.88 ppm . Analytical data are consistent with those previously reported in the literatureTo a solution of palmitic acid  in ice-cold dichloromethane (12.5 ml), triethylamine , HOBt  and TBTU  were added sequentially, and the mixture was stirred for 30 min. Then methylamine hydrochloride  was added to the reaction, that was stirred overnight (synthetic schemes are collected in the Supporting Information). The mixture was then diluted with dichloromethane and washed with HCl 1 M, then saturated NaHCOS)-2-amino-1-propanol  was added to the reaction, that was stirred for 2 more hours (synthetic schemes are collected in the Supporting Information). The mixture was then diluted with dichloromethane and washed with HCl 1 M, then saturated NaHCO3, and brined. The organic phase was dried over MgSO4, and the crude product was recrystallised from heptane to get 100 mg of target product (yield = 48%). MS calc.: 313.30, found: 314.3 m/z [M + H]+. To a solution of palmitic acid  in ice-cold dichloromethane (10 ml), triethylamine , HOBt  and TBTU  were added sequentially, and the mixture was stirred for 30 min. Then : δ 5.53 ppm , 4.07 , 3.67 ppm , 3.54 ppm , 2.74 , 2.18 ppm , 1.63 ppm , 1.38–1.20 ppm , 1.17 , 0.88 ppm . Analytical data are consistent with those previously reported in the literatureR)-2-amino-1-propanol  was added to the reaction, that was stirred for 2 more hours (synthetic schemes are collected in the Supporting Information). The mixture was then diluted with dichloromethane and washed with HCl 1 M, then saturated NaHCO3, and brined. The organic phase was dried over MgSO4, and the crude product was recrystallised from heptane to get 106 mg of target product (yield = 57%). MS calc.: 313.30, found: 314.3 m/z [M + H]+. 1H-NMR : δ 5.54 ppm , 4.07 , 3.67 ppm , 3.54 ppm , 2.75 , 2.18 ppm , 1.63 ppm , 1.38–1.20 ppm , 1.17 , 0.88 ppm . Analytical data are consistent with those previously reported in the literatureTo a solution of palmitic acid  in ice-cold dichloromethane (9 ml), triethylamine , HOBt  and TBTU  were added sequentially, and the mixture was stirred for 30 min. Then (ATGATGATGATGATGATGGCTTGGGTTTCTGATC (XhoI restriction site is underlined). A (6x) Histidine tag was introduced in the reverse primer sequence (highlighted in bold). The PCR product was cloned into pCDNA 3.1 vector (Invitrogen) using HindIII and XhoI restriction enzymes (New England Biolabs). HEK-293 cells (American Type Culture Collection) were cultured at 37 °C in an incubator (5% CO2) using complete Dulbecco’s modified Eagle’s medium (DMEM) containing 10% foetal bovine serum (FBS), 1% penicillin-streptomycin and 1% L-glutamine, and transfected with the rat NAAA(6xHis)-pCDNA3.1 construct using JetPEI transfection reagent (Polyplus), following manufacturer’s instructions. Stably transfected cells were selected by addition of G418 (1 mg mL−1) to the cell culture medium and cell clones were generated by limiting dilution plating. Growing clones were analysed for NAAA expression by western blot .The encoding sequence of rat NAAA (BC105771) was amplified from the cDNA clone 7930474 (Open Biosystems) using the following primer pair: forward primer 5′- CCCAAGCTTATGGGGACCCCAGCCATCCG (HindIII restriction site is underlined); reverse primer 5′-CGCTCGAGTCACell pellets were resuspended in Tris-HCl buffer (50 mM) containing sucrose . Samples were sonicated with a Branson digital sonifier 102 C, and centrifuged at 800 g for 15 min at 4 °C. The resulting supernatants were centrifuged at 12,000 g for 30 min at 4 °C. The pellets were suspended in phosphate-buffered saline  and subjected to two freeze/thaw cycles at −80 °C. The suspensions were centrifuged at 100,000 g for 1 h at 4 °C. Protein concentrations were measured using BCA analysis, and samples were stored at −80 °C until use.Z-10-heptadecenoic acid  or heptadecanoic acid  as an internal standard. Samples were centrifuged , and the organic phases were transferred into new vials, dried and the residues were dissolved in 0.3 ml of methanol:chloroform . Fatty acid quantifications were carried out by LC/MS using an Agilent 6410 Triple Quad LC/MS system, and the analytes were eluted from a Zorbax Eclipse XBD-C18 column  at a flow rate of 0.4 ml min−1 for 6 min using a solvent mixture of water (A) and methanol (B), both containing 0.25% acetic acid and 5 mM ammonium acetate . Column temperature was set at 40 °C. Electrospray ionisation was in the negative mode and the capillary voltage was 4 kV. N2 was used as drying gas at a flow rate of 13 L min−1 and a temperature of 350 °C. The [M−H]− ion was monitored in the selected-ion monitoring (SIM) mode. Calibration curves were generated using commercial fatty acids . NAAA activity was calculated as pmol of fatty acid produced by 1 mg of NAAA in 1 min. Activity data were plotted as function of the substrate concentration, and Km and Vmax values were calculated using the Michaelis-Menten function analysis on GraphPad Prism version 8.4.2 (GraphPad Software Inc.).Substrates were incubated, in a range from 1 to 200 µM, in 100 mM Sodium Phosphate Monobasic, 100 mM Sodium Citrate, 0.1% Triton-X 100, 3 mM DTT, pH 4.5 (NAAA assay buffer) with 4 µg of NAAA at 37 °C for 30 min (triplicate samples). Reactions were terminated by addition of 0.6 ml of a cold mixture of methanol and chloroform (1:1) containing Z configuration located at the Δ9 or Δ10 position  exhibited lower Vmax and higher Km values, relative to PEA , 10-fold higher than C14 (2) and 5-fold higher than C15 (3). A similar effect was seen for FAEs with longer acyl chains, i.e. C17 (5) and C18 (6) (6), stearic acid, was only detectable at substrate concentrations equal or higher than 100 µM. The results, summarised in 4, C16) and sharply declines when the fatty acyl chain is either shortened  or lengthened .FAE species with C13, C14 and C15 acyl chains (compounds  C18 (6) . The hydcis double bond might affect the efficiency of NAAA-mediated FAE hydrolysis. We prepared and tested FAE species with acyl chains C15:1Δ10 (7), C16:1Δ9 (8), C17:1Δ10 (9), C18:1Δ9 , C19:1Δ10 (11) and C20:1Δ9 (12)  and longer-chain compounds (10–12) including OEA (max/Km ratios compared to PEA (We next asked whether the introduction of a 1Δ9 (12) . FAE C171Δ9 (12)  and dispding OEA . Of noted to PEA .i) a hydrogen atom, yielding the primary amide 13; ii) a methylamine group, yielding the secondary amide 14; or iii) a 2-amino-1-propanol group in the (S) or (R) configuration . All the substrates were hydrolysed by NAAA, albeit with different Vmax and Km  in complex with a molecule of myristic acid (C14:0). The selection of the template structure was driven by the fact that myristic acid is a natural product of NAAA catalysis and is thus unlikely to cause significant perturbations to the co-crystallised lipophilic pocket, compared to the perturbation expected for reversible or irreversible inhibitors such as ARN19702 and ARN726To investigate the molecular basis of the substrate recognition and specificity of rNAAA, we built a homology model of the enzyme starting from the available crystal structure of the rabbit form of the enzyme  with its carboxylic group in neutral state and engaged in an H-bond with the neutral form of the enzyme’s We submitted the rNAAA-PEA complex to a 200 ns molecular dynamics simulation in order to assess its dynamic stability. The analysis of root mean squared deviation (RMSD) of the protein backbone and of the heavy atoms of residues that shape the active site revealed that the overall geometry of the model is conserved, with the RMSD fluctuating around an average value of 1.08 Å. A similar value was recorded in a simulation performed starting from the template structure of the crystallised ocNAAA-myristic acid complex . RMSD an1 formed an unstable complex in which the amide group easily moved away from the active site, losing the possibility to engage the carbonyl carbon and the sulphur atom of Cys131 in interactions that would favour the nucleophilic attack  or Δ10 (compound 9), did not affect the binding mode of these substrates, which similarly to what observed for compounds 1–6 placed the amide group within the active site, taking H-bonds with the oxyanion hole residues and the backbone of Asp150. The position of the cis double bond of the three compounds slightly overlapped, fitting the same portion of the hydrophobic channel occupied by the piperazine fragment of ARN19702 in the X-ray structure of hNAAA (PDB: 6DXX.pdb)9, that carries the unsaturation at position Δ10, was characterised by a higher stability compared to 8 and 10. Nonetheless, compound 9 could not maintain the initial conformation, with the amide group inserted within the active site, for more than 10 ns , and compared the results with an analogous simulation on the NAAA-PEA complex. The simulations showed that both 15 and 16, similarly to PEA, are characterised by high stability, with the amide fragment steadily maintained within the active site and the carbonyl carbon at a distance from the sulphur atom of Cys131 oscillating around an average value of ≈3.3 Å, which is consistent with a nucleophilic attack. While the values recorded indicated that all the three substrates maintain a pro-reactive accommodation within the active site, they did not account for the higher propensity of 15 to be hydrolysed by NAAA relative to its R-enantiomer and PEA. For this reason, we analysed the MD simulations focussing on the behaviour of the hydroxyl group of the polar head in the three substrates. The plots reported in N-terminal amino group of Cys131 (15, compared to PEA and 16. An additional polar interaction between the hydroxyl group and the terminal amino group of Cys131 was also observed for PEA and 15, with conformations having distances shorter than 3 Å more populated for the latter.To investigate the effect of modifications at the ethanolamide portion, and specifically of the replacement of the hydroxyl group with a bulkier fragment, we performed a set of 200 ns MD simulations of the complexes between NAAA and f Cys131 , measurem and Vmax values, and used the Vmax/Km ratio as a parameter to evaluate catalytic efficiency. The results of kinetic experiments were rationalised by evaluating the dynamic stability of the complexes between the substrates and a model of rNAAA.In this study, we combined biochemical and molecular modelling approaches to delineate a detailed outline of NAAA’s substrate selectivity. This is critical to define the precise contribution of this enzyme to lipid-amide signalling and may be helpful in the design of better inhibitors. Starting from a library of potential substrates, we determined the Kmax/Km ratio of substrates with slightly different acyl chains suggests that the 16 carbon-atom saturated acyl chain of PEA is a critical feature for the recognition of the substrate and its hydrolysis. This substrate selectivity mirrors the peculiar shape of NAAA’s binding site, characterised by a straight and narrow hydrophobic channel. The correlation between the acyl chain length and the catalytic efficiency was also reflected by the results of molecular modelling studies. MD simulations results suggest that NAAA selectively recognises substrates able to fit within its narrow and straight binding pocket, while substrates with a non-optimal acyl chain form unstable complexes in which the amide group is not allowed to take the interactions that would favour the nucleophilic attack. Lastly, we investigated the role of the ethanolamine group by testing palmitamide, N-methylpalmitamide, and R- and S-methyl PEA. Kinetic experiments show that derivatives lacking the ethanolamine portion are rapidly hydrolysed by NAAA, with a Km comparable to that measured for PEA. Consistently with these data, we recently reported the results of theoretical studies on the mechanism of NAAA showing that the acylation step modelled for PEA and N-methylpalmitamide is characterised by a similar free-energy profileR- and S-methyl PEA, which showed Km values comparable to PEA, also pointed out that substrates with relatively bulky polar heads can be accommodated within the active site of the enzyme. MD simulations suggest that substitution in the α position of the ethanolamine group promotes a conformational selection in the substrates, promoting an enrichment in conformations that can make polar contacts within the active site of the enzyme for the S enantiomer. Collectively, the results of our biochemical and molecular modelling studies outline the fundamental role of the hydrophobic tail of substrates in the recognition process by NAAA, suggesting that the optimisation of a hydrophobic portion able to mimic the conformation of the 16-carbon saturated tail of PEA would be a critical step in the design of novel site-active NAAA inhibitors.Our results show the existence of a strict correlation between the catalytic efficiency of the enzyme and the length of its substrate acyl chain, with an optimum for PEA. A steep fall in the VClick here for additional data file.