Our findings indicate that several acetylation sites in tau are responsive to HDAC6 and that acetylation on Lys-321 (within a KCGS motif) is both essential for acetylation-mediated inhibition of tau aggregation in vitro and a molecular tactic for preventing phosphorylation on the downstream Ser-324 residue. To determine the functional consequence of this HDAC6-regulated phosphorylation event, we examined tau's ability to promote microtubule assembly and found that phosphorylation of Ser-324 interferes with the normal microtubule-stabilizing function of tau. Tau phosphorylation of Ser-324 (pSer-324) has not previously been evaluated in the context of tauopathy, and here we observed increased deposition of pSer-324–positive tau both in mouse models of tauopathy and in patients with Alzheimer's disease. These findings uncover a novel acetylation–phosphorylation switch at Lys-321/Ser-324 that coordinately regulates tau polymerization and function. Because the disease relevance of this finding is evident, additional studies are needed to examine the role of pSer-324 in tau pathobiology and to determine whether therapeutically modulating this acetylation–phosphorylation switch affects disease progression in vivo.The aberrant accumulation of tau protein is a pathological hallmark of a class of neurodegenerative diseases known as tauopathies, including Alzheimer's disease and related dementias. On the basis of previous observations that tau is a direct substrate of histone deacetylase 6 (HDAC6), we sought to map all HDAC6-responsive sites in tau and determine how acetylation in a site-specific manner affects tau's biophysical properties  O-GlcNAc modifications O-GlcNAcylation; and serine/threonine/tyrosine phosphorylation  that prevents tau filament assembly tylation . To do sts in AD . In addits in AD . Of partin vitro (in vitro acetylation reaction of recombinant full-length tau (4R0N isoform) by co-incubating with acetyl-CoA and p300, followed by the addition of recombinant HDAC6 to the reaction in the presence or absence of an HDAC6 inhibitor (ACY-738). As an additional control, we included a reaction in which recombinant tau was incubated with acetyl-CoA in the absence of the p300 acetyltransferase enzyme to control for potential nonenzymatic acetylation and/or autoacetylation of tau that may occur . To identify acetylated residues, samples were separated by SDS-PAGE and subsequently visualized by silver staining . Bands corresponding to the molecular weight of tau were excised and evaluated by mass spectrometry (MS) following digestion. Utilizing two different MS analysis techniques, we determined the sites modified by acetylation, with residues numbered according to the longest human tau isoform (441 amino acids)  . Acetylao acids) . Of noteitor ACY-38. As anXGS motifs (K259Q/K290Q/K321Q/K353Q (4KQ)) significantly impaired tau polymerization (a). Given that K290Q/K321Q (2KQ) also decreased tau aggregation, acetylation of tau's internal KCGS motifs appears to be most critical to inhibit tau filament assembly. To further assess the extent to which acetylation at each KXGS motif impacts tau assembly, we generated mutant proteins containing a single mutation, revealing that K321Q dramatically inhibits tau filament formation (a). Of note, whereas mimicking acetylation at most non-KXGS sites had minimal impact on tau aggregation, K274Q also significantly decreased tau assembly in vitro, albeit to a lesser extent than K321Q (b).To dissect the contribution of tau's various acetylation sites to the ability of tau to assemble into filaments, we generated recombinant proteins containing lysine-to-glutamine mutations to mimic acetylation. As observed previously, mimicking acetylation on all four of tau's Krization a. Given ormation a. Of notan K321Q b.a, c, and d–f) and a and b)supplemental Fig. S2 , K321Q dramatically alters tau filament assembly and aggregation (a and b and d–f) and a and b)supplemental Fig. S2 . In particular, despite the reduced absolute number of filaments formed by K321Q tau (e and f), some long filaments were infrequently detected following polymerization of K321Q (b and d), which might indicate that acetylation of Lys-321 partially inhibits the initial nucleation but not elongation of filaments.As an alternative approach to evaluate the impact of Lys-321 acetylation on tau polymerization, we used pelleting analysis and electron microscopy to examine tau filament formation. Whereas mutating Lys-321 to arginine to maintain the charge but prevent acetylation has no significant impact on filament formation compared with wild-type tau . We then monitored phosphorylation of KXGS motifs in HEK293T cells co-transfected with either WT or K321Q tau in the presence or absence of MARK2. As anticipated, WT tau was phosphorylated at both Ser-324 (pSer-324) and Ser-262/356 (12E8) when co-expressed with MARK2 . However, the pseudoacetylated K321Q mutant was resistant to MARK2-mediated phosphorylation at Ser-324, whereas phosphorylation at the 12E8 epitope was unaffected . These findings indicate that similar to KIGS motifs, the ability of MARK2 to phosphorylate KCGS motifs is regulated by acetylation.We previously observed that acetylation of KIGS motifs (Lys-259/353) prevented phosphorylation by MARK2 at the corresponding serine residues (Ser-262/356) . To deteFig. 3c). Given that phosphorylation of Ser-262 in tau's first KIGS motif has also been shown to impair tau's ability to promote microtubule assembly  or prevent (S324A) phosphorylation at Ser-324 and subsequently measured the ability to promote microtubule assembly. Whereas both WT and S324A tau were capable of supporting the polymerization of microtubules, the pseudophosphorylated S324D and S324E mutants exhibited an impaired capacity to drive microtubule assembly c. Given assembly , the phosupplemental Fig. S3), we assessed whether pSer-324–positive tau species were detected along with other phospho-tau epitopes in our recently developed AAV model of tauopathy (a–f). Furthermore, although pSer-324 is absent from rTg4510 mice at 2 months of age, pSer-324–positive tau is detected in rTg4510 mice at both 6 and 10 months of age . These findings demonstrate that pathological pSer-324 deposition is observed in mouse models of tauopathy, and the lack of detection at early stages of disease may suggest that pSer-324 positivity is indicative of a more advanced stage of pathology.Whereas deposition of tau species phosphorylated on KIGS motifs (pSer-262/356) increases with age in the well-characterized rTg4510 mouse model , 12, it auopathy . At 6 moauopathy . At thisauopathy , a–f. Fua and supplemental Fig. S5). The presence of pSer-324–positive tau histologically also differentiates AD cases (c–e) from control subjects , collectively indicating that phosphorylation of tau on Ser-324 does not occur under normal conditions and is associated with disease.Phosphorylation of tau on KIGS motifs (pSer-262/356) is observed at early stages of neurofibrillary tangle (NFT) formation in AD , but assFig. 5m ACY-738 for 24 h to inhibit HDAC6 activity. Cell lysates were then collected, and tau levels and phosphorylation were evaluated by immunoblotting . Consistent with previous studies (a and c) and supplemental Fig. S6). We also observed a striking decrease in phosphorylation at Ser-324, which was statistically significant even when normalizing to E1 to control for the reduction in tau levels (a and b) and supplemental Fig. S6). In the absence of a site-specific antibody to detect acetylation of tau at Lys-321, we measured acetylation of tubulin as an indication of HDAC6 inhibition (a and d) and supplemental Fig. S6). These findings indicate that accumulation of pSer-324–positive tau species is sensitive to HDAC6 activity in neurons.To determine whether inhibition of HDAC6 in neurons impacts the accumulation of tau species that are hyperphosphorylated on Ser-324, we transduced mouse primary neuronal cultures with human P301L tau-AAV and treated them with 1 μ studies , 16, HDAIn the current study, we have identified acetylation of Lys-321 as a critical modification for acetylation-mediated inhibition of tau aggregation. In addition to impeding tau filament assembly, acetylation of Lys-321 also prevents phosphorylation on Ser-324, a phosphorylation event that we show is detrimental to tau function. Establishing disease relevance, we confirm that phosphorylation of tau on Ser-324 is detected in two different mouse models of tauopathy as well as in patients with AD. Given that a recent study also identified phosphorylation of Ser-324 (Ser-313 in endogenous mouse tau) in transgenic mice expressing human amyloid precursor protein, but not nontransgenic control mice , phosphoA recent report utilizing cryo-electron microscopy to investigate the structure of straight filaments and paired helical filaments (PHFs) purified from AD brain now provides novel insight into the role of Lys-321 in regulating filament formation . SpecifiXGS acetylation and block KXGS phosphorylation. Although in contrast to our observations that tau acetylation with the acetyltransferase p300 impairs filament assembly, acetylation of tau with the acetyltransferase CBP has been previously reported to either increase  but was identified under conditions that reduced aggregation  was detected in nontransgenic mice  has been shown to be required for tau toxicity in osophila . In addiosophila , 26, are2/356 hasosophila , 27–29, phila 27–, 31. AltXGS motifs can also be modified by ubiquitination and methylation . Because acetylation of Lys-321 also inhibits phosphorylation on Ser-324, and the aberrant accumulation of pSer-324–positive tau is detected in APP and tau animal models, as well as patients with AD, augmenting acetylation of Lys-321 would be anticipated to prevent both tau aggregation and abnormal phosphorylation of Ser-324. Therefore, additional studies are needed to examine the role of pSer-324 in tau pathobiology as well as to determine whether therapeutically modulating the acetylation–phosphorylation switch at Lys-321/Ser-324 impacts disease progression in vivo.In conclusion, our findings indicate that tau acetylation on Lys-321, which is detected in nontransgenic mice , signifid-galactopyranoside from EMD Millipore .ACY-738 was synthesized and kindly provided by Acetylon Pharmaceuticals, Inc. . We purchased the recombinant proteins p300 , HDAC6 , and tubulin . We also purchased phosphatase inhibitor mixtures II and III, nicotinamide, trichostatin A, DNase, acetyl-CoA, dextran sulfate , thioflavin S, Coomassie Blue, and β-mercaptoethanol from Sigma-Aldrich. In addition, we purchased DTT (Fisher), protease inhibitor mixture, and isopropyl 1-thio-The rabbit anti-ac-KIGS antibody (ac-Lys-259/353) was generated and affinity-purified by 21st Century Biochemicals , as described . E1 hum, 35, 36.MARK2/Par1-myc was kindly provided by B. Lu , whereas GFP and wild-type V5-tagged tau (4R0N isoform) constructs for mammalian expression were cloned into pcDNA3.1 in our laboratory , 36. TheA600 reached 0.5 absorbance units. Tau expression was then induced with 0.5 mm isopropyl 1-thio-d-galactopyranoside for 4 h, and the cells were collected by centrifugation and washed in 1× TBS before storing pellets at −80 °C.Human cDNAs encoding either WT or mutant tau (4R0N isoform) were subcloned into pET-30a bacterial expression vectors  and transformed in Rosetta 2(DE3) pLysS competent cells (EMD Millipore) for expression. Cultures were grown to saturation overnight and were subsequently utilized to inoculate larger cultures in the morning (1:100 dilution). Cells were grown in Luria Broth on a shaker  until the m Tris (pH 8), 50 mm NaCl, 1 mm MgCl2, 1 mm PMSF, 1 mm dithiothreitol, 21 units/ml DNase) and lysed with three freeze-thaw cycles using liquid nitrogen and a tepid water bath. The lysates were then centrifuged to eliminate cell debris, and 500 mm NaCl was added to supernatants, which were heated  and subsequently cooled on ice (10 min). Precipitates were removed by centrifugation, and the resulting supernatant was loaded onto a 5-ml HiTrap Mono S column  using the ÄKTA FPLC system . The column was washed with 10 column volumes of wash buffer (10 mm HEPES (pH 7.4), 150 mm NaCl), and tau proteins were eluted with a linear gradient of NaCl at 20 mm/min. Fractions of ∼1 ml were collected and analyzed by SDS-PAGE and Coomassie Blue staining. Fractions that contained high concentrations of purified tau protein were then pooled and dialyzed against 200 volumes of 10 mm HEPES buffer at pH 7.4. A BCA protein assay kit (Pierce) was then used to determine protein concentration.For purification, the cell pellets were first resuspended in lysis buffer (100 mm recombinant tau was incubated with 0.5 μg of recombinant human p300 in buffer A (10 mm HEPES (pH 7.4), 50 mm NaCl, 1.5 mm MgCl2, 0.5 mm dithiothreitol, 2.5 mm EGTA, 0.1 mm EDTA) with 125 μm acetyl-CoA for 3 h at 30 °C . Following the acetylation reaction, 1 μg of recombinant human HDAC6 in the presence or absence of ACY-738 (10 μm) was added to the reactions as indicated and incubated at 37 °C for 2 h.To acetylate tau, 8 μin vitro acetylation and deacetylation of recombinant tau, reactions were immediately separated by SDS-PAGE. One gel was transferred to PVDF membrane to confirm tau acetylation/deacetylation by immunoblotting, whereas the second gel was stained with the Pierce Silver Stain Kit. The bands corresponding to tau were excised and subsequently submitted for mass spectrometry analysis by the Medical Genome Facility Proteomics Core at Mayo Clinic. Given the abundance of lysine residues and the potential for a high abundance of short peptides that could result from digestion with trypsin, the bands were cut in half, and half of each band was digested with trypsin, whereas the remainder was digested with chymotrypsin. The trypsin and chymotrypsin digests from each reaction were then pooled into a single tube, and the four reactions  were subsequently run on the ThermoScientific Orbitrap Elite mass spectrometer. The data were collected using two different MS/MS fragmentation methods, including higher-energy collisional dissociation and scanning the fragments into the orbitrap or scanning with the ion trap using collision-induced dissociation. The higher resolving power of the orbitrap higher-energy collisional dissociation scans provided accurate mass fragments, whereas the collision-induced dissociation ion trap scanning is faster and more sensitive. The database search was performed allowing for acetyl-Lys and protein N-terminal acetylation.Following the m HEPES (pH 7.4), 50 mm NaCl, 1.5 mm MgCl2, 0.5 mm dithiothreitol, 2.5 mm EGTA, 0.1 mm EDTA) at a concentration of 8 μm (0.33 mg/ml) was polymerized by incubating with 0.04 mg/ml dextran sulfate at 37 °C for 4 h. Thioflavin S was then added to the reaction to a final concentration of 7.5 μm, and 30 min later, tau filament assembly was monitored by fluorescence of thioflavin S binding using a Cary Varian Eclipse spectrofluorometer . Thioflavin S binding intensity was measured by integrating the curve in the range of 460–600 nm using the Cary Eclipse Scan software.Recombinant tau solubilized in buffer A  for 45 s. These were then stained with 2% uranyl acetate (Electron Microscopy Sciences) for 45 s, and the grids were examined with a Philips 208S electron microscope . To quantify filament formation, four images were collected randomly at three predetermined coordinate locations that remained fixed throughout these studies. These 12 images were collected at ×5,000 magnification, and the average filament number, average filament length, and total filament length per field were measured manually using ImageJ freeware. The length bar was also measured so that these lengths could be converted into nanometers.g for 75 min at 4 °C to separate soluble and insoluble, aggregated tau. The pellets containing aggregated and polymerized tau were resuspended in 30 μl of 1× SDS sample buffer, separated by SDS-PAGE on a 10% Tris-glycine gel, and subsequently visualized by staining with Coomassie Blue.Following the polymerization reaction, samples were centrifuged at 100,000 × HEK293T cells were maintained in Opti-MEM  supplemented with 10% heat-inactivated FBS (Life Technologies) and 1% penicillin/streptomycin (Life Technologies) passaged every 3–4 days based on 90% confluence. For plasmid transfections, 2 μg of plasmid DNA  was combined with Lipofectamine 2000 reagent (Life Technologies) for 20 min in 500 μl of Opti-MEM (serum-free medium), and this mixture was added to cells for 4 h. The transfection mixture was then replaced with fresh complete medium, and cells were harvested 24 h posttransfection.m) was added to an equal volume of recombinant tau  in assembly buffer  to a final reaction volume of 100 μl and incubated at 37 °C. The amount of tubulin assembly was determined by turbidity assay per the manufacturer's recommendation. The absorbance was measured every 15 s for a duration of 30 min at 340 nm on SpectraMax M5 Multi-Mode microplate readers . Reactions were performed in triplicate, and both the rate and extent of microtubule polymerization were assessed.The ability of recombinant WT and mutant Ser-324 tau proteins to promote microtubule assembly was evaluated in a 96-well format. Ice-cold tubulin at a concentration of 3.0 mg/ml (60 μm Tris-HCl (pH 7.4), 274 mm NaCl, 5 mm KCl, 5 mm EDTA, 1% Triton X-100, 1% SDS, 1 mm PMSF, protease inhibitor mixture, and phosphatase inhibitor mixtures II and III), followed by sonication. Samples were centrifuged at 16,000 × g for 15 min at 4 °C, and a standard BCA protein assay (Pierce) was performed on the supernatant. Cell lysate (30 μg of protein) was diluted in distilled H2O, 2× Tris-glycine SDS sample buffer (Life Technologies), and 5% β-mercaptoethanol (Sigma-Aldrich) and heat-denatured for 5 min at 95 °C. Samples were run on SDS-PAGE Tris-glycine gels (Life Technologies) and transferred to PVDF membrane (Millipore). Membranes were blocked in 5% nonfat dry milk in TBS, 0.1% Triton X-100 and incubated overnight in primary antibody rocking at 4 °C. Membranes were incubated in HRP-conjugated secondary antibodies  for 1 h at room temperature and detected by ECL (PerkinElmer Life Sciences). Bands were quantified using Scion Image by analyzing pixel density, and protein levels were normalized to GAPDH as the protein loading control.Cells were lysed in homogenate buffer . For the aging study, male rTg4510 mice were aged to the indicated time points and euthanized by cervical dislocation, and their brains were quickly removed, hemisected, and frozen on dry ice. The right forebrain from both rTg4510 and P301L tau-AAV mice (model described in Ref. g at 4 °C) to remove cellular debris. Supernatants were collected, protein concentration was determined by BCA assay, and samples were processed as described above for immunoblotting.Human post-mortem brain tissue provided by the brain bank at Mayo Clinic Jacksonville included frontal cortex samples from six control patients  and six AD patients . The average age of control patients was 86 years, whereas the average age of AD patients was 70 years . The frozen brain samples were weighed and homogenized in a 10× volume of homogenate buffer, followed by sonication and centrifugation  for 30 min, and endogenous peroxidase activity was blocked by incubation in 0.03% hydrogen peroxide. Sections were then immunostained using the DAKO Autostainer  and the DAKO EnVision + HRP system. The stained slides were then dehydrated, coverslipped, and scanned with the Aperio slide scanner .TM A medium without calcium (BrainBits), supplemented with B27 (Life Technologies), 0.5 mm GMAX (Life Technologies), and gentamicin (Life Technologies) until all dissections were complete. Corticohippocampal tissue was then digested in papain , triturated with a Pasteur pipette (bore size 0.8–1 mm), centrifuged to collect cell pellet, and resuspended in Neurobasal A (Life Technologies), supplemented with B27, GMAX, gentamicin, and basic fibroblast growth factor (Life Technologies). Following determination of cell number, neurons were seeded at a density of 3 × 105 cells/well on poly-d-lysine–coated 6-well plates. Medium was changed on day in vitro (DIV) 3 and 7, and AAV was added to cultures on DIV 4. ACY-738 (1 μm) was added on DIV 9, and cultures were harvested on DIV 10.For primary neuronal cultures, cortex and hippocampi from postnatal day 1 mouse pups were removed and briefly stored in HIBERNATEt test. p < 0.05 was considered statistically significant.GraphPad Prism was utilized to perform statistical analyses. Differences among multiple groups were analyzed using one-way analysis of variance, with post hoc analysis using Dunnett's multiple-comparison tests. Differences between vehicle– and ACY-738–treated neurons  were assY. C., L. P., and C. C. designed the research. Y. C., D. C. C., M. Y., and M. C.-C. performed the research. B. J. M. performed mass spectrometry analysis. J. D. and J. T. provided technical assistance. M. D. performed electron microscopy. Y. C., M. D., D. W. D., L. P., and C. C. analyzed and interpreted the data. L. P. and C. C. wrote the article, and all authors reviewed and approved the final version of the manuscript.


O-glucoside, apigenin, and (Z) and (E)−2-hydroxy-4-methoxycinnamic acid glucosides as the active (poly)phenols in Chamomile (Matricaria recutita) able to modulate carbohydrate digestion and absorption in vitro as assessed by inhibition of α-amylase and maltase activities. The latter two compounds previously mistakenly identified as ferulic acid hexosides were purified and characterised and studied for their contribution to the overall bioactivity of chamomile. Molecular docking studies revealed that apigenin and cinnamic acids present totally different poses in the active site of human α-amylase. In differentiated Caco-2/TC7 cell monolayers, apigenin-7-O-glucoside and apigenin strongly inhibited D-[U-14C]-glucose and D-[U-14C]-sucrose transport, and less effectively D-[U-14C]-fructose transport. Inhibition of D-[U-14C]-glucose transport by apigenin was stronger under Na+-depleted conditions, suggesting interaction with the GLUT2 transporter. Competitive binding studies with molecular probes indicate apigenin interacts primarily at the exofacial-binding site of GLUT2. Taken together, the individual components of Chamomile are promising agents for regulating carbohydrate digestion and sugar absorption at the site of the gastrointestinal tract.Transient hyperglycaemia is a risk factor for type 2 diabetes and endothelial dysfunction, especially in subjects with impaired glucose tolerance. Nutritional interventions and strategies for controlling postprandial overshoot of blood sugars are considered key in preventing progress to the disease state. We have identified apigenin-7- Gastrointestinal side-effects of acarbose have resulted in poor patient compliance and adherence to the treatment, highlighting the need for the identification of new molecules which could restrict post-prandial burden. To this end, we previously screened several plant extracts and identified German chamomile (Matricaria recutita) (ChE) as very effective at attenuating carbohydrate digestion and sugar absorption in vitro3.Excess consumption of carbohydrates and sugars can lead to hyperglycaemia, endothelial dysfunction and insulin resistance, increasing the risk of developing of type 2 diabetes and cardiovascular diseases. The modulation of carbohydrate digestion in the intestine by the non-absorbed drug acarbose reduced the risk of type 2 diabetes and cardiovascular events by 25 and 49% respectively in a population with impaired glucose tolerant according to The Study To Prevent Non-Insulin dependent Diabetes Mellitus trial4. It can potentially relieve the metabolic overload effects of high sugar diets such as glucose and fructose by attenuating their absorption through the inhibition of carbohydrate-digestive enzymes and apically-located transporters3; this mechanism may explain some of the positive effects observed by chamomile in experimental diabetes6 and type 2 diabetics8.Chamomile is used as an herbal tea all over the world, and recognition of its medicinal properties is exemplified by its listing as an official drug in the pharmacopoeias of 26 countries including the UK10. (Poly)phenol content varies depending on growth conditions and extraction methods and this can have a pronounced impact on biological activities. Therefore, a first and essential step is to identify the specific active constituents responsible for the bioactivity. In another study where authors attempted to identify inhibitors of digestive enzymes in chamomile a convincing single candidate was not described5. Furthermore, individual components of chamomile have been previously reported to have a beneficial inhibitory effect on monosaccharide transport; however, the contribution of each on the overall exerted effect by ChE is not known. Here we report the inhibitory activity of the main (poly)phenol constituents of ChE on the different steps of carbohydrate digestion and absorption, while for the first time we have fully confirmed and characterized two previously misidentified glycosylated hydroxycinnamic acids present in ChE at high concentrations and assessed their contribution to the potential of ChE to counteract hyperglycaemia.Chamomile contains many different phytochemicals, mainly (poly)phenols, a class of small molecules with several bioactivities3 where we identified by mass spectrometry apigenin, apigenin-7-O-glucoside and two compounds with corresponding [M-H]- ion masses at m/z 355 and 711 (compounds 1 and 2) as the most abundant constituents. In this study, apigenin-7-O-glucoside (compound 3) and apigenin (compound 4)  based on the total ion count chromatograms (TIC).Initial detection of target (poly)phenols in ChE was based on our previous report 4) Fig. ; Table 1 4) Fig.  were cha1H and 13C NMR, NOESY and chemical synthesis. The full-scan TOF spectra of both compounds showed prominent ions at m/z 355.1035 and 711.2136, which were assigned as [M-H]- and [2M-H]- respectively  elemental formula of the two hydroxycinnamic acid derivatives was established as C16H20O9. Both compounds showed identical fragment ions in the Q/TOF product ion spectra at m/z 193.0506 [M-hexosyl-H]- and 149.0608 [M-hexosyl-CO2]-. This result excluded the possibility that either of the compounds is a dimer of ferulic acid hexoside as suggested by Guimarães et al.11 based on the predominant ion at m/z 711 for compound 2. Covalently bound ‘dimeric ferulic acids’ are well characterised compounds, which act like polysaccharide cross-linking agents in cell walls13, and therefore their (di)glycoside structures can also be theoretically expected in plant extracts. However, the predicted ion formula [C32H37O18]- would result in a monoisotopic ion mass of m/z 709.1985 in negative ion mode. Confirmatory fragmentation of the m/z 711.2136 was performed by Q/TOF that provided fragments only at m/z 355 and 193, lending further support to our original assumption that the elemental formula of the two hydroxycinnamic acid derivatives is C16H20O9. Further analysis was performed to conclusively rule out the isomeric structures of ferulic acid hexosides previously suggested.15The identities of the hydroxycinnamic acid derivatives were assigned based on HPLC-DAD/QTOFMS, m/z(-) 193 (supplementary S2) with UV spectra characteristic of hydroxycinnamates was detected at 13.4 min  and (E)-alkene group in the structure of compound 1 and 2 were identified from the signals at δ 7.19 and 5.78 (J = 12.7 Hz), and δH 7.82 and 6.39 (J = 16.2 Hz), respectively. The spectrum also indicated the presence of three aromatic protons in the ABD substitution pattern characteristic of the coupling constants of J = 8.5–8.6 (ortho) and J = 2.4–2.5 (meta)17. Acid hydrolysis of compounds 1 and 2 resulted in the release of D-glucose  and 4.98 (E-isomer) with coupling constants of J = 7.5 Hz from the anomeric protons indicate a β-glycosidic linkage and the location of the D-glucose moiety was assigned at the aromatic ring by the observed NOE enhancements  and (E)−2-β-D-glucopyranosyloxy-4-methoxycinnamic acid (Z and-E-MCAG). Further confirmation of the identity of the cinnamate moiety was obtained by chemical synthesis, and comparison with the released aglycone after acid hydrolysis -MCAG were also active, although they showed only modest inhibition with maximum values ~25 and ~20% respectively (p < 0.05)   Fig. . When th05) Fig. . Moreovep < 0.05) inhibition was evident. When individual constituents were tested at concentrations equivalent to those present in ChE and higher, rat maltase was similarly only weakly inhibited by the individual (poly)phenols and apigenin 7-O-glucoside was the principal contributor (p < 0.05) -MCAG was resistant to digestive hydrolytic enzymes, indicating that 2-(E)-MCA would not be produced in significant amounts in the small intestine, and therefore 2-(E)-MCA was not assessed for inhibition of carbohydrate-digesting enzymes (supplementary S3).While ChE weakly inhibited rat intestinal maltase, no inhibitory effect was observed on rat sucrase and isomaltase Fig. ; even at05) Fig. . Neverth05) Fig. . None of197, (b) the formation π-π stacking interactions with Trp59 and Tyr62 and (c) the stacking towards the binding subsites −1 and −2. As shown in supplementary S4, these observations were extended to other strong (poly)phenol inhibitors of human α-amylase (arbitrarily defined as molecules with IC50 <100 µM) including EGCG, quercetin and luteolin18. Apigenin 7-O-glucoside is able to form π-π stacking interactions with Trp59 and Tyr62, however it does not form the H-bond with the catalytic residue Asp197 resulting in a ~10 fold lower inhibition in agreement with the in vitro results phenols. Based on the strong inhibition observed for apigenin, three important features were identified: (1) the formation of H-bonds between the OH group of the B ring and the catalytic residue Asplts Fig. .Z) and (E)-MCAG bind in a completely different pose -MCAG, an H-bond is formed between the 6-OH group of the glucose unit with Asp197 while (E)-MCAG presents a more complex interaction. In (E)-MCAG, 2 covalent bonds are formed between the carbon at position 2 and Asp197 -isomer  is active, in addition to the facilitated transporters GLUT2 and GLUT5 phenols, apigenin 7-O-glucoside was a very effective inhibitor of D-[U-14C]-glucose and D-[U-14C]-sucrose transport, and IC50 values were obtained  and (E)-MCAG had no effect on D-[U-14C]-glucose, D-[U-14C]-sucrose or D-[U-14C]-fructose transport (data not shown). The combination of apigenin and apigenin 7-O-glucoside when tested at concentrations equivalent to those quantified in the extract, accounted for ~62, 80 and 89% of the total transport inhibition of D-[U-14C]-glucose, D-[U-14C]-sucrose and D-[U-14C]-fructose, respectively, exhibited by ChE phenols in ChE were responsible for the effect, each was tested separately on D--glucose transport experiments under Na+-free conditions where only GLUT2, and not SGLT1, is active. An apparent increase (~50%) in the inhibitory activity was observed for ChE (p < 0.05), apigenin 7-O-glucoside and apigenin as evidenced by the decrease in the inhibition constants, suggesting that the main inhibitory effect is on GLUT2   Fig. .O-glucoside. Immunostaining of GLUT2 showed that GLUT2 protein was present on both apical and basolateral sides of differentiated Caco-2/TC7 cells  confirming that apigenin 7-O-glucoside can interact directly with GLUT2 transporter protein.Glycosylated (poly)phenols can be hydrolysed by luminal β-glucosidase enzymes, such as lactase phlorizin hydrolase24. Delaying the absorption of sugars in the intestine is considered a clinically relevant approach for reducing type 2 diabetes risk23. The present study was designed to identify the (poly)phenols involved in the inhibition of carbohydrate digestion and absorption previously demonstrated by ChE3 and further explore their mechanism of action.Repeated high postprandial glucose spikes and fructose related metabolic burden have been proposed as independent risk factors for developing insulin resistance and progression to type 2 diabetesZ) and (E)−2-β-D-glucopyranosyloxy-4-methoxycinnamic acids in ChE, in agreement with the tentative identification of Weber et al.17. These compounds were previously misidentified as ferulic acid hexosides and ‘dimer’ forms by several authors25. When they were individually tested, they showed modest inhibitory activity on human α-amylase; however, when assessed in combination with apigenin and apigenin 7-O-glucoside, it was evident that they significantly contribute to the overall bioactivity of ChE.In the work presented here we have provided conclusive evidence of the presence of  its catalytic activity26. Unlike apigenin 7-O-glucoside, apigenin showed additional interaction with Asp197 through an H-bond and this was reflected in a ~10-fold increase in its inhibitory activity. Thus, H-bonds between OH groups of the B ring and catalytic residues, and π-π stacking interactions appear to be requirements for an optimal inhibition of human α-amylase by (poly)phenols acting via a competitive mechanism. Indeed, in a previous study, Lo Piparo et al.27 observed similar features; (poly)phenols showing IC50 <100 µM were anchored in the active site through π-π interactions with Trp59, and formed H-bonds with residues Asp197 and Glu233 through OH groups in the A and B rings. This mechanism is further supported by X-ray structural analyses of the (poly)phenols myricetin and montbretin A with human pancreatic α-amylase29.30 while sucrase-isomaltase additionally hydrolyses sucrose, producing glucose and fructose. Inhibition of α-glucosidases is complementary in strategies aiming at blunting blood sugar spikes stemming from consumption of readily absorbable sugars which increase the prospect of type 2 diabetes.32 Our results indicate only a modest inhibition of maltase-glucoamylase by the four (poly)phenols, even when tested in combination, consistent with the inhibition observed for ChE. In contrast, Kato et al.5 found a stronger inhibition of ChE against maltase-glucoamylase (IC50 = 2.6 mg/mL) and sucrase-isomaltase (IC50 = 0.9 mg/mL) using brush border membranes prepared from rat small intestine. The authors suggested that this effect was driven by the presence of esculetin  and quercetin aglycone . However, the phytochemical composition and content of the plant material used in the study was not reported. Data from other studies on chamomile composition indicate that these compounds, if present at all, are unlikely to be effective since they are several orders of magnitude below the concentrations necessary for inhibition34.The final step of starch digestion is catalysed by α-glucosidases, maltase-glucoamylase and sucrase-isomaltase. Their combined catalytic activities hydrolyse linear and branched malto-oligosaccharides after the action of pancreatic α-amylase36 Our data show that apigenin and apigenin 7-O-glucoside are potent and selective inhibitors of GLUT2 as these compounds account for up to ~80% of transport inhibition under Na+-depleted conditions. They bind at the exofacial site of the protein as demonstrated by comparing binding affinities with specific inhibitors of the intra- and extracellular cavities of the transport channel. In contrast, apigenin and apigenin 7-O-glucoside showed a modest inhibition of fructose transport and only a small fraction of the transport of D-[U-14C]-fructose was inhibited following hydrolysis of D-[U-14C]-sucrose by brush-border α-glucosidase. However, a higher inhibition of fructose transport in a physiological context can be expected since the apical expression of GLUT5 in the Caco-2 clone is comparable to the one noted in an adult small intestine37 and Caco-2/TC7 cell line presents a 2-fold increase in apical GLUT5 compared to Caco-2 cells3.At low concentrations, the uptake of glucose and fructose relies on SGLT1 and the facilitated transporter GLUT5, respectively. However, after consumption of a meal high in sugars, glucose concentration may reach 50–300 mM in the intestinal lumen prompting the facilitated GLUT2 transporter to translocate to the apical membrane. GLUT2 accounts for ~75 and ~60% of total glucose and fructose uptake respectively.O-glucoside are bioavailable in humans but are rapidly metabolised and excreted, while they are subject to extensive colonic catabolism39. Currently, there are no pharmacokinetic data on (Z)- or (E)-MCAG but it is anticipated that they can reach the colon and are subject to catabolic reactions as observed for other hydroxycinnamic acids40, especially given that (E)-MCAG was not a substrate for rat intestinal enzymes. Based on information from other related compounds no harmful effects are predicted after dietary exposure of these (poly)phenols through consumption.Like other (poly)phenols, apigenin and apigenin 7-p-nitrophenyl-glucopyranoside, rather than the physiological substrate 43. Many studies have used yeast as a source of α-glucosidase activity, which has no relevance to the mammalian enzymes44. Hence apigenin did not inhibit yeast α-glucosidase action on the non-physiological substrate p-nitrophenyl-glucopyranoside in one report45, but did inhibit in other reports with an IC50 value of 22 μM in46 and of 25.5 μg/ml in47. In the latter report, apigenin-7-O-glucoside also inhibited with an IC50 value of 29.7 μg/ml47. Apigenin lowered GLUT1 expression in a nude mouse model of laryngeal carcinoma48 and in prostate cancer cells49.Several papers have reported that apigenin in various herbal and plant extracts can inhibit α-glucosidase activity. For example, apigenin-7-O-glucoside inhibited rat intestinal α-glucosidase activity, but the study used Z) and (E)-MCAG, apigenin and apigenin 7-O-glucoside, could pose a good alternative to modulate postprandial sugar excursions. Unlike the antidiabetic drug acarbose, the combination of these (poly)phenols can mildly attenuate carbohydrate digestion and sugar absorption, avoiding the gastrointestinal side effects of acarbose. Furthermore, apigenin and apigenin 7-O-glucoside could be employed either alone or in combination with other inhibitors for an enhanced formulation, for example in a population that has already developed a pathophysiological phenotype.Based on our data, we propose that ChE, a natural source that combines  equipped with an Gemini C6 phenyl column . The ChE extract (150 mg/mL) was loaded manually (1 mL) using a syringe through a sample loop of 1 mL and eluted using water containing 0.1% TFA (solvent A) and methanol (Solvent B) at a flow rate of 2.3 mL/min as follows: 0–12.8 min linear gradient to 24% B; 12.8–25.6 min isocratic at 24% B; 25.6–111 min linear gradient to 100% B; 111–123.8 min isocratic at 100%B, 123.8–125 min linear gradient to 5% B: 125–150.6 min isocratic at 5%B. The elution was followed at 320 nm and fractions containing the separated compounds (supplementary S1) were collected and analysed for purity by LC-MS (supplementary S6). Multiple semi-preparative separation cycles were undertaken and the fractions for each peak combined, freeze-dried, and stored at −20 °C.m/z 50–1100. Next, manually selected ion peaks of the TOF-only spectra were subjected to Q/TOFMS analysis and elemental formulae of fragments were determined based on accurate mass (<20 ppm) data obtained from product ion peak spectra.Analysis of the purified hydroxycinnamic acid derivatives was carried out using an Agilent 1200 series HPLC system coupled to an Agilent 6530 Q/TOF mass spectrometer  equipped with a dual spray ESI source operating in negative mode. Followed by an isocratic separation with reversed phase HPLC, elemental formulae of compounds were determined with the aid of MassHunter software based on high-resolution  accurate mass (<5 ppm) data completed with evaluation of isotope abundance matching and isotope spacing. Data was obtained from full-scan TOF-only peak spectra acquired in the mass range of An aliquot of the purified compound 2  and UV detection at 250 nm. 10 µL of sample was eluted with 0.1 mM ammonium acetate buffer (pH 5.5)–acetonitrile  at 1 mL/min. The identification of the glycosyl component was done by comparing the retention time with those of authentic standards .A second aliquot was subjected to hydrolysis, concentrated to dryness and derivatised with 1-phenyl-3-methyl-5-pyrazolone (PMP) as described elsewhere6 DMSO and transferred to 5 mm OD NMR tubes. All NMR data were recorded on a Bruker Avance 500 spectrometer  temperature controlled at 299.3 K. Chemical shifts are expressed in ppm and are reported with reference to the residual solvent peak. Multiplicities are reported with coupling constants and are given to the nearest 0.1 Hz. Where needed, two-dimensional correlation spectroscopy (2D-COSY), heteronuclear single quantum coherence spectroscopy (HSQC) and heteronuclear multiple bond correlation spectroscopy (HMBC) and nuclear Overhauser effect spectroscopy (NOESY) was performed.Purified samples were dissolved in dZ)-2-D-Glucopyranosyloxy-4-methoxycinnamic acid present as a mixture of E and Z in a 1:3 ratio, E peaks eliminated and Z peaks picked at: 1H NMR  δ 7.70 , 7.19 , 6.74 , 6.56 , 5.78 , 4.85 , 3.77 , 3.72 , 3.47 , 3.38 , 3.34–3.29 *, 3.16   δ 167.7, 161.0, 160.7, 156.7, 136.32, 131.1, 117.53, 116.73, 101.11, 100.5, 77.24, 76.86, 73.27, 69.81, 60.72, 55.33. *Overlayed by strong signal from water. ESI-MS: m/z 355.1035 [M-H]-. The 1H assignments in the chemical structure are shown in supplementary S7-A.(E)−2-β-D-Glucopyranosyloxy-4-methoxycinnamic acid: 1H NMR  δ 12.00 , 7.82 , 7.62 , 6.76 , 6.62 , 6.39 , 5.16 ), 4.98 , 4.89 ), 4.58 ), 3.78 , 3.72 , 3.47 , 3.38 , 3.34–3.29 *, 3.16 . 13C NMR  δ 168.08, 162.08, 157.07, 138.76, 129.32, 116.73, 116.05, 108.03, 101.11, 100.02, 77.24, 76.86, 73.27, 69.81, 60.72, 55.33. *Overlayed by strong signal from water. ESI-MS: m/z 355.1035 [M-H]-. The 1H assignments in the chemical structure are shown in supplementary S7-B. NOESY spectrum and key enhancements are shown in supplementary S8 and Fig. Analysis of pure (trans carboxylate by NMR in D2O. The solution was then acidified with 1 M aqueous HCl and extracted with dichloromethane (3 × 10 mL). The solvent was then evaporated and eluted with ethyl acetate on a flash silica column. This yielded the (E)−2-hydroxy-4-methoxycinnamic acid (E-MCA) . The low yield was due to the ring closing to reform the coumarin in acidic conditions. 1H NMR  δ 9.25–9.21 , 7.95 , 7.54 , 6.40 , 6.37  6.33 , 3.66 . The 1H assignments in the chemical structure are shown in supplementary S7-C.7-Methoxy coumarin  was dissolved in an aqueous solution of 0.5 M NaOH (20 mL) and stirred for 4 h at room temperature. Aliquots were taken and showed full conversion to the m/z 193.06 [M-H]-(supplementary S9).The synthesised compound was analysed by HPLC-DAD as described in section 2.3. ESI-MS: 52. The ChE stock solution was dissolved in DMSO while the individual (poly)phenols were prepared in a water-EtOH solution . The maximum concentration of DMSO and EtOH in the assay was 2 and 1% respectively, which did not affect the activity of the enzyme as assessed in control incubations.The human α-amylase and rat α-glucosidase inhibition assays were performed under optimised conditions as previously reported53. The α-amylase structure was prepared for docking by removing from the crystallographic structure water molecules and co-crystallised ligands. No constraints were introduced and the default parameters of the dock module of the CLC Drug Discovery 3.0.1 (CLC Bio-Qiagen) were used. Validation of the docking parameters was performed by docking the co-crystal modified structure of acarbose into the active site. A root mean square deviation of 1.36 Å was obtained between the original and the docked conformations (supplementary S10). The number of docking iterations for evaluation of the preferred binding pose was set at 500. Ten best docked poses per docking calculation were analysed based on their docking score and the top ranked pose for each ligand was selected for further analysis. Ligand-receptor interactions were visualised with CLC workbench and Discovery Studio .We investigated the interactions of (poly)phenols with human α-amylase using the three-dimensional crystal structure 1MFV3.Caco-2/TC7 cells were cultured and transport experiments performed as fully described before4 cells/well on 12 well Millicell cell culture inserts  and maintained for 21–23 d at 37 °C (10% CO2). For staining, cells were fixed with 4% para-formaldehyde in PBS and incubated with wheat germ agglutinin  for 10 min at 37 °C. Following permeabilisation with 0.1% Triton-X100 for 20 min at room temperature, cells were incubated with GLUT2 antibody  at a dilution of 1:50 for 1 h, or without primary antibody for controls. After washing with PBS, cells were further incubated with Cy3-conjugated donkey anti-rabbit IgG at a 1:300 dilution. Cell layers were stained with 2 μg/mL 4′−6-diamidino-2-phenylindole (DAPI) and mounted on microscope slides with ProLong Gold antifade reagent mounting medium  and imaged using a Zeiss LSM700 confocal microscope with a Plan-Apochromat 63 × oil immersion objective.Caco-2/TC7 cells were seeded at 7 × 105 cells/well on 6-well plates  and cultured for 21–23 d at 37 °C as described above. Cells were exposed to 150 μM of apigenin 7-O-glucoside for 25 and 60 min. After the treatment the medium was collected and analysed by HPLC as detailed above.Cells were seeded at 2.4 × 10t-test was used to compare means of the contribution of apigenin 7-O-glucoside and apigenin to the total inhibition of sugar transport. Data are expressed as the mean ± standard error of mean (SEM).Statistical analysis was performed by one-way ANOVA using the Number Cruncher Statistical System version 6.0 software . Significant differences were assessed with Tukey-Kramer multiple comparison test (p < 0.05). Independent samples Data available on the Open Science Framework (ID: osf.io./fs32u).Supplementary figures


There have been few studies regarding physical training‐induced peripheral nerve dysfunction in patients with hereditary neuropathy with liability to pressure palsies (HNPP), with the exception of soldiers that trained intensively. Here, we report a 15‐year‐old boy without family history of HNPP who developed bilateral painless brachial plexopathy following short‐term barbell and plank training during a school baseball club activity. Muscle training‐induced painless brachial plexopathy could be an initial symptom and may be underdiagnosed in adolescents with sporadic HNPP. The patient also had unapparent leg‐muscle weakness. Mild sensory disturbance of superficial sensation was detected in the left forearm. The tendon reflex in the upper extremities was diffusely reduced, but that in the legs was preserved. Long tract signs were unapparent. The results of blood and cerebrospinal fluid (CSF) analyses, including serum antiganglioside antibodies and CSF protein, were unremarkable. Cervical magnetic resonance (MR) imaging did not detect any spinal cord lesions; however, maximum intensity projection images on coronal T2‐weighted imaging  showed enlargement and hyperintensity of the bilateral brachial plexuses, especially on the left side Figure  and/or tde novo onset (Infante et al., HNPP is an autosomal dominant genetic disease; however, 20%–78% of patients constitute sporadic cases with subclinical asymptomatic relatives and To date, few patients with HNPP that developed brachial plexopathy after physical training have been reported (Horowitz et al., The case presented here is the first case of an adolescent with HNPP that developed neurological symptoms during muscle training in a school baseball club activity. In addition, the patient developed brachial plexopathy after barbell and plank training, neither of which has been previously reported as a trigger of HNPP. In our patient, the barbell would have compressed the brachial plexus directly, which may have induced left side plexopathy similar to backpack carriage (Horowitz et al., To the best of our knowledge, there has been only one case of HNPP that was evaluated using MR plexography (Chhabra et al., Our case indicated that muscle training‐induced brachial plexopathy could be an initial symptom and may be underdiagnosed in adolescents with HNPP. Patients with HNPP might need to focus on particular muscle training exercises, such as barbell lifting, planks, and push‐ups, as repetitive causative training could induce irreversible axonal degeneration (Horowitz et al.,


Viscum album) products used in cancer therapy are frequently combined with other anti-cancer-drugs. Hence, potential herb-drug interactions have become a major safety concern in mistletoe therapy.European mistletoe  were tested for inhibition of nine major cytochrome P450 (CYP) isoenzymes in a test system using pooled human liver microsomes and for induction of five CYP isoforms in human hepatocytes cultivated in vitro according to the relevant guideline.Major inhibition did not occur in any of the CYP marker reactions. For some CYP isoenzymes, a minor or intermediate inhibition could be observed, but without dose effect relationship. Induction activity (≥ 1.5-fold increase) was not found with any of the three mistletoe products.Since no induction capacity was found and major inhibition above 50% did not occur even with the highest concentration used, which is approximately 100,000-fold higher than the clinically relevant dose in plasma, a clinically relevant herb-drug interaction is not expected for Helixor® A, M, and P. Viscum album belong to the most frequently used herbal medicines in cancer patients in Central Europe, especially in Germany [Hypericum perforatum) to chemotherapy with irinotecan not only deceased therapy-related myelosuppression, but also decreased the plasma levels of the active metabolite of the anti-cancer drug [European mistletoe products for injection made of  Germany . During  Germany . Phase I Germany . A numbe Germany  indicaticer drug . Thus, tcer drug . The resw/v) aqueous summer and winter extracts from fresh mistletoe herb . The three different sorts  represent three different host trees: Helixor® A from fir , Helixor® M from apple tree  and Helixor® P from pine . There are clear differences between these three Helixor® sorts in the content and spectrum of various active constituents, e.g. mistletoe lectins, and also in cytotoxic activity [Three solutions for injection, produced by Helixor Heilmittel GmbH, Rosenfeld, Germany, were tested . All products are manufactured from 5%  were purchased from In Vitro Technologies, Inc. Baltimore, USA as pooled, mixed gender human liver microsomes, art. no. X00821, lot no. MGU, delivered on dry ice and stored at −80 °C until use.Freshly isolated, pre-plated human primary hepatocytes from five different donors  were supplied by Cytonet GmbH & Co. KG, Weinheim, Germany and used for the enzyme induction assays.7-Ethoxyresorufin, content 99.0 / 100%, lot. No. 021 K4024 (inhibition test) and 033 K4058 (induction test), Sigma; coumarin, content 99.9%, lot no. 90 K3684, Sigma; S-mephenytoin, content 99.0% lot no. P6178, Biomol; paclitaxel, content 99.3%, lot. No. 3508F, ICN; diclofenac sodium, content >99%, lot. No. 107H4748, Sigma; bufuralol hydrochloride salt, content 99.5%, lot. No. 102–155, Ultrafine; chlorzoxazone, content 99.6%, lot. No. S08663–121, Aldrich; testosterone, content 99.9%, lot. No. S542, Applichem.Furafylline, purity 98.9%, lot. No. 063 K4702, Sigma; 8-methoxypsoralene, purity >98%, lot no. 30122, ICN; triethylenethiophosphoramide, purity 100.9%, lot no. 081 K1195, Sigma; ketoconazole purity 99.3%, lot. No. 78353, ICN; sulfaphenazole purity 99.1%, lot. No. 3G03777, Applichem; omeprazole, purity 100%, lot. No. 021 K1605, Sigma; quinidine sulphate, purity 99.3%, lot. No. 4353B, ICN; diethyldithiocarbamic acid sodium salt, purity 100%, lot. No. 110 K2615, Sigma.Omeprazole, purity 100%, lot no. 021 K1605, Sigma; phenobarbital sodium, purity 99.0%, lot no. 076H0292, Sigma, ethanol, purity 99.9%, lot no. 3006761, AppliChem, rifampicin, purity 99.6%, lot no. 304324/1, Fluka.m. The test items and reference inhibitors were preincubated with microsomal suspension for 5–10 min  or 11.5–12.5 min , respectively, and the incubations were started by addition of the respective marker substrate. The incubation was stopped after 30 ± 1 min by addition of stop reagent (150 μl 100% acetonitrile or 50 μl 43% phosphoric acid for CYP2E1), centrifuged for 10 min at 4 °C and approximately 3000 x g, and the supernatant was transferred to a new 96-well microtiter plate for marker reaction specific analysis of remaining marker substrates and resulting metabolites.The mistletoe products were tested for effects on marker reactions specific for CYP 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. Final concentrations of 0.5, 0.005, and 0.0005 mg/ml were used. Incubation was carried out in 96-well microtiter plates at 37 ± 0.5 °C in the presence of 2 mM NADPH and 3.3 mM magnesium chloride with pooled human liver microsomes  in 50 mM potassium phosphate buffer, pH 7.4. All tests were performed in triplicate. The total volume of the individual incubation was 200 μl. The marker substrate concentrations in the incubation mixtures were chosen at concentrations close to their apparent KMarker reactions containing no test item and no CYP specific inhibitor but identical concentrations of the corresponding solvent type of the test item incubations served as negative controls of inhibition (NC). Marker reactions containing a marker reaction specific reference inhibitor instead of one of test items served as positive controls of inhibition (PC).The final concentrations of the marker substrates in the respective incubation media were as follows: 5 μM 7-ethoxyresorufin for CYP1A2; 5 μM coumarin for CYP2A6, 200 μM S-mephenytoin for CYP2B6; 10 μM paclitaxel for CYP2C8; 10 μM diclofenac for CYP2C9; 50 μM S-mephenytoin for CYP2C19; 20 μM bufuralol for CYP2D6, 50 μM chlorzoxazone for CYP2E1; 100 μM testosterone for CYP3A4.The measured substrate specific metabolites were resorufin for CYP1A2; umbelliferone for CYP2A6; nirvanol for CYP2B6; 6α-hydroxy-paclitaxel for CYP2C8; 4′-hydroxy-diclofenac for CYP2C9; 4′-hydroxy-mephenytoin for CYP2C19; hydroxybufuralol for CYP2D6; 6′-hydroxy-chlorzoxazone for CYP2E1; 6β-hydroxy-testosterone for CYP3A4.The following final concentrations of CYP specific inhibitors in the incubation samples were used: 25 μM furafylline for CYP1A2; 0.75 μM 8-methoxypsoralen for CYP2A6; 75 μM triethylenethiophosparamide for CYP2B6; 25 μM ketoconazole for CYP2C8; 2 μM sulfaphenazole for CYP2C9; 7.5 μM omeprazole for CYP2C19; 0.3 μM quinidine for CYP2D6; 30 μM diethyldithiocarbamic acid sodium salt for CYP2E1; 0.15 μM ketoconazole for CYP3A4.5 cells were plated on collagen-coated 24-well plates in medium optimized for hepatocytes (HIM) containing 100 IU/ml penicillin, 100 μg/ml streptomycin and 0.05 μMol dexamethasone. After addition of test item or reference inducer, plates were cultivated for 48 ± 4 h under the following incubation conditions: 37 ± 2 °C, 5% CO2, approximately 95% humidity. Three incubation mixtures were prepared per concentration per test item, positive and negative control per CYP isoenzyme. Medium (containing test reagent where applicable) was changed once after 24 ± 2 h. All wells were checked by microscopic analysis for contamination and cell morphology before incubation with test item, positive / negative controls and at the end of incubation. After the induction phase the supernatants were removed and replaced by medium supplemented with 3 mM salicylamide (S-HIM). After 10 min incubation at 37 °C the supernatants were replaced by 350 μl/well S-HIM containing the respective concentration of the specific substrate for the enzyme marker reactions. After further 3 h ± 15 min of incubation the supernatants of all samples were collected in 2 ml tubes which contained 350 μl ice-cold 100% acetonitrile or 70 μl of 43% phosphoric acid for the CYP2E1 incubation. Precipitated protein was sedimented by centrifugation for 10 min at 4 °C and approximately 14,000 rpm. 200 μl of the supernatants were transferred to microtiter plates for marker reaction specific quantification of remaining marker substrates and resulting metabolites.For evaluation of CYP induction activities of the mistletoe products, freshly isolated human primary hepatocytes from three different donors were used. 2–5 × 10Helixor® A, M and P were added at final concentrations of 10 μg/ml, 5 μg/ml and 0.5 μg/ml each. Hepatocyte incubations containing no test item but the respective reference inducers served as positive controls (PC). The following final concentrations were used: 50 μM omeprazole for CYP1A2; 1 mM phenobarbital sodium for CYP2B6 and CYP2C9; 100 mM ethanol for CYP2E1; 10 μM rifampicin for CYP3A4. Incubations without test item or reference inducers in medium containing 0.5% water served as negative controls (NC) for incubations with Helixor® A, M and P.The final concentrations of the marker substrates in the respective incubation media were as follows: 2 μM 7-ethoxyresorufin for CYP1A2; 1 mM S-mephenytoin for CYP2B6 (100/200 μM for the first three donors. Since metabolite concentration was below level of quantification the higher concentration was used for the following donors); 50 μM diclofenac sodium for CYP 2C9; 200 μM chrorzoxazone for CYP 2E1; 200 μM testosterone for CYP 3A4.The marker metabolite of CYP 1A2 (resorufin) was quantified by fluorescence based sample analysis  using a Fluoroskan Ascent microplate fluorometer .For the assessment of effects on the CYP isoenzymes 2B6, 2C9, 2C19, 2E1, and 3A4 (induction or inhibition) as well as 2A6, 2C8 and 2D6 (inhibition only) the respective marker substrates and marker metabolites were separated and detected by use of HPLC  and mass spectroscopy  on a C18 column (Luna 3u C18(2), 30 × 2.0 mm, 3 μm, Phenomenex Aschaffenburg, Germany). For CYP 2E1 a C8 column (Luna C8 (2), 150 × 2.0 μm, 5 μm) was used. Further HPLC parameters are summarized in Table Data analysis of the CYP induction experiments was performed using standard software: MS-EXCEL™ , MassLynx™ V3.5and QuanLynx™ , Ascent Software V2.4.2 , and ChemStation Rev.A.09.01 . The mean of n replicates, standard error of mean, % compared to negative control, and standard error were calculated using the respective Excel-functions.In those cases where fluorescence was detected using a micrometer plate reader, the blank was substracted from the means before further calculations. Based on the means the percentage peak areas or fluorescence units compared to the negative control were derived. Additionally to standard blank samples, a test item interference control sample (blank sample containing the highest test item concentration but without the respective marker substrates) was used for fluorescence analysis in order to detect effects exhibited by the test item possibly interfering with the fluorescence based sample analysis.In the incubation experiments with human microsomes the mean and the standard error of mean of the signals  of the replicate incubation per test item concentration or per control at the end of the incubation time were determined as measures of the relative activity of the respective test system.The relative standard error of mean of the qualifier peak areas should be less than 20%. In those cases, where sample sequences contained less than four qualifiers, the difference between the lowest and highest qualifier values were evaluated and accepted if the difference was less than 30%. Otherwise the analysis of this series was repeated. In the case of the CYP inhibition experiments the data was accepted only if the positive control of inhibition inhibited the respective marker reaction at least 30% compared to the respective negative control of inhibition.The definition of major, intermediate and minor inhibition was an increase of marker reaction activity of >50%, 25–50% and <25%, respectively; the definition for induction was a ≥ 1.5-fold increase in marker reaction activity compared to the negative control.Under the conditions used in this study, none of the test items exhibited major inhibition (above 50%) of any of the CYP marker reactions. Intermediate inhibition (from 25 to 50%) was observed for Helixor® A with CYP2A6 and CYP2C9 Fig. , for HelHepatocytes incubated with Helixor® A demonstrated no changes in cell morphology compared to negative control. Incubation with Helixor® M at 10 μg/ml and with Helixor® P at 5 μg/ml and 10 μg/ml led to minor changes in cell morphology of hepatocytes from some donors. Change in morphology was accompanied with a partial detachment of cells from substrate and is likely due to a moderate cytotoxic effect exerted by Helixor® A and P at the highest concentrations.The criterion for an induction of the tested CYP isoenzymes (an at least 1.5-fold increase in marker reaction activity compared to the negative control) was not met by any of the test items at any of the concentrations for all cytochromes P450 isoenzymes tested Table . All tesThe aim of the studies was to investigate the in vitro potential for induction or inhibition by the three commercially available mistletoe products Helixor® A, M and P of clinically relevant cytochrome P450 isoenzymes.Induction of CYP marker reactions was tested in freshly isolated human hepatocytes with respect to the CYP isoenzymes 1A2, 2B6, 2C9, 2E1, and 3A4. At the highest concentration (10 μg/ml) Helixor® P and partly Helixor® M showed a moderate cytotoxic effect on hepatocytes of some donors. In this respect, 10 μg/ml was the highest concentration employable in the induction studies with the test items, which is approximately 2000-fold higher than the clinically relevant dose in plasma.The results of the marker reaction experiments indicated no induction potential of all the Helixor® products on the five CYPs investigated at any of the concentrations tested. Marker reaction activity in hepatocytes incubated with the test items was in the range of the negative control for all donors and all CYPs tested.The experiments on inhibitory effects of Helixor® A, M and P on the enzymatic activity of nine CYP isoforms in human liver microsomes yielded no major inhibition (above 50%) with any of the CYP marker reactions, even with the highest concentration of 500 μg/ml. Furthermore, for the CYP isoenzymes either minor or intermediate inhibition could be observed but with no dose effect relationship. Putative increase as well as decrease of inhibition of the corresponding marker reactions with increasing concentrations of test item was observed for various CYPs. Thus, the mentioned putative dose dependent effects are most likely due to statistical variations of approximately 20% and cannot be regarded as inhibition. It has also to be considered that inhibition rates of approximately 20% are likely irrelevant in a biological (in vivo) system.These findings regarding inhibitory and stimulatory effects of the Helixor® products confirm and extend the results of another in vitro study on the capacity of Helixor® M and three other mistletoe products to induce the metabolic activity in the liver cell line HepG2 . For evaThe results presented here are also in agreement with the results of very similar investigations on three other mistletoe products . These pSilybum marianum) and grapefruit juice for example both proved to inhibit CYP3A4 isoenzyme in vitro [Hypericum perforatum) decreased the plasma levels of the active metabolite of irinotecan indicating stimulation rather than inhibition of CYP3A4 and other major metabolism pathways [Hypericum on CYP3A4 and other CYP isoenzymes [In fact, demonstration of significant effects of herbal compounds on CYP isoenzymes in vitro usually causes concerns about interactions of those drugs with cytostatic therapies. However, such concerns often cannot be confirmed in vivo. Milk thistle  [This conclusion is supported by the results of several clinical studies. A phase I trial investigated the impact of an additional therapy with Helixor® A to gemcitabine in patients with far advanced tumors . In thisIn a randomized controlled trial the effects of an additional therapy with the mistletoe products Helixor® or Iscador® during chemotherapy were assessed at the end of the treatment showing significantly better tolerance of chemotherapy and an increase in quality of life in the verum groups as compared to the untreated controls , 21. TheFinally, in a non interventional study gynecological outdoor patients were interviewed with a standardized questionnaire on use of CAM. The authors concluded that interactions of mistletoe products with conventional drugs are unlikely when used in addition to chemotherapy or endocrine therapy. Only in case of antibodies the risk for interactions with mistletoe therapy was classified possible due to theoretical considerations while it was stated that no data regarding direct interactions or interactions via metabolism by the cytochrome P450 systems are available . MeanwhiThese results corroborate the assumption that better tolerance of chemotherapy and putatively also of other oncological treatments due to additional mistletoe treatment may not be accompanied by a decreased anti-tumor efficiency of the conventional therapies as there is no evidence for pharmacologically and clinically relevant interactions.For the clinically most relevant CYP isoenzymes 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4, no induction capacity (i.e. an at least 1.5-fold increase of marker reaction activity) was found. Furthermore, no major inhibition above 50% of marker reaction activity occurred even with the highest concentration of the mistletoe products, which is approximately 100,000-fold higher than the therapeutic dose in plasma. Thus, a clinically relevant herb-drug interaction involving CYP isoenzymes is not to be expected for Helixor® A, M, and P.


Plasmodium falciparum causes malaria in humans with over 450,000 deaths annually. The asexual blood stage involves invasion of erythrocytes by merozoites, in which they grow and divide to release daughter merozoites, which in turn invade new erythrocytes perpetuating the cycle responsible for malaria. A key step in merozoite invasion is the essential binding of PfRh5/CyRPA/PfRipr complex to basigin, a step linked to the formation of a pore between merozoites and erythrocytes. We show CyRPA interacts directly with PfRh5. An invasion inhibitory monoclonal antibody to CyRPA blocks binding of CyRPA to PfRh5 and complex formation thus illuminating the molecular mechanism for inhibition of parasite growth. We determined the crystal structures of CyRPA alone and in complex with an antibody Fab fragment. CyRPA has a six-bladed β-propeller fold, and we identify the region that interacts with PfRh5. This functionally conserved epitope is a potential target for vaccines against P. falciparum.DOI:http://dx.doi.org/10.7554/eLife.21347.001 P. falciparum with approximately 214 million cases and over 450,000 deaths each year occurring mostly in subtropical and tropical regions of the world . The inP. falciparum Rh5 interacting protein (PfRipr)  and (PfRipr) . The fun(PfRipr) . Furthernhibited . The funost cell . RegardlCyRPA and PfRipr are localized in the micronemes whereas PfRh5 is present at the neck of the rhoptries and these proteins are released onto the surface during merozoite invasion . Super-rP. falciparum in a NOD-scid IL2Rynull mice engrafted with human erythrocytes  was expressed in insect cells and purified to homogeneity as evidenced by size-exclusion chromatography (SEC) and SDS-PAGE analysis . Whilst P. falciparum growth was determined. Three monoclonal antibodies  were identified that inhibited growth to approximately 70% at 2 mg/ml, whereas others (7A6 and 8B9) showed essentially no inhibition . The structure of the complex was determined using diffraction data to 2.44 Å resolution by molecular replacement using a homologous Fab fragment to obtain initial phases, followed by iterative model building and crystallographic refinement . Crystalvia the formation of a β-sandwich comprising sheet 6–6 of one CyRPA dimer and sheet 6–6 of the second CyRPA dimer , with bacterial sialidase (neuraminidase) from Arthrobacter ureafaciens as a positive control  is relatively high for antibody-antigen complexes  approximately perpendicular to the barrel axis of the 6BBP assembly. The Fab fragment of 8A7 omplexes , with thomplexes . The intomplexes .10.7554/P. falciparum into human erythrocytes through binding the receptor basigin  the antibody conformational epitope, defined by residues within the loop between the second and third strands of the first β-sheet, the loop between the first and second β-sheets, and residues at the N-terminus region of the fourth strand of the second β-sheet. The absence of large conformational change within CyRPA upon Fab complexation rules out an allosteric mechanism of abrogating a complex formation with PfRh5. Therefore, blocking of the CyRPA and PfRh5 interaction by antibodies is consistent with a functional PfRh5/CyRPA/PfRipr complex assembling at the interface between the invading merozoite and erythrocyte membrane  full-length mature CyRPA with the last residues of the three potential glycosylation site motifs mutated to alanine  was cloned in to the vector pgpHFT  of GenScript (New Jersey). Amplified antibody fragments were cloned separately into a standard cloning vector separately. Colony PCR was performed to screen for clones with inserts of correct sizes. No fewer than five colonies with inserts of correct sizes were sequenced for each fragment. The sequences of different clones were aligned and the consensus sequence of these clones was obtained.P. falciparum growth in vitro. 8A7 was used for co-crystallography studies and is isotype IgG1 kappa. Non-inhibitory anti-PfRh5 monoclonal antibody 4G4 and inhibitory anti-PfRipr monoclonal antibody 5G6 were included as controls.All procedures with mice were approved by the Walter and Eliza Hall Medical Research Institute Animal Ethics Committee. BALB/c mice were immunized with 20 µg of recombinant CyRPA in Freund's complete adjuvant  followed by two subsequent injections of 20 µg CyRPA in Freund's incomplete adjuvant  then 40 µg CyRPA in saline and 50 µg CyRPA in saline. Mice were euthanized and spleens harvested using aseptic technique. Spleen cells were fused with myeloma cells. Anti-CyRPA antibody-producing hybridoma cell lines were identified 14 days after fusion, using ELISA with recombinant CyRPA. Positive hybridomas were sub-cloned to obtain monoclonal cell lines. Positive antibodies were subsequently screened by immuno-blot with mature schizonts. Monoclonal antibodies 7A6, 8B9, 3D1, 8A7 and 5B12 were selected for further study and tested to determine if they inhibit P. falciparum, as described to determine if the monoclonal antibodies inhibited parasite growth  at a parasitaemia of 0.1%. Parasitaemia was counted after 96 hr by flow cytometry and specific growth inhibition calculated relative to parasites grown in non-immune IgG. P. falciparum strain 3D7 has been confirmed by whole genome sequencing and was originally obtained from Prof. David Walliker, Edinburgh University.Two cycle growth inhibition assays were performed, using 3D7 strain of e growth . Serial bis-Tris precast NuPAGE gel  then transferred to a nitrocellulose membrane. The membranes were probed with the anti-CyRPA monoclonal antibodies (1/1000) and anti-HA mouse monoclonal (1/1000) as a positive control and proteins detected by enhanced chemiluminescence .Synchronized schizont stage 3D7 parasites that expressed HA-tagged CyRPA  were harSEC was performed on an analytical Superdex 200 column or Superose-6 column  and proteins were eluted with 25 mM HEPES, 150 mM NaCl, pH 7.2 or 25 mM Tris, 150 mM NaCl, pH 8.5. Co-immunoprecipitation of CyRPA with FLAG-tagged PfRh5 was performed using anti-FLAG M2 agarose (Sigma). Briefly, 5 µg FLAG-tagged PfRh5 and 5 µg CyRPA were incubated with 20 µl anti-FLAG M2 agarose beads for 1 hr on ice. After extensive washing with 25 mM Tris, pH 8.5 buffer containing 150 mM NaCl, the bound proteins were eluted with the FLAG peptide at a concentration of 100 μg/ml in the same Tris buffer, analysed by SDS-PAGE and stained with Coomassie Blue R250.Crystallization trials for both the Fab-bound and the uncomplexed CyRPA protein were initially performed in sitting-drop, 96-well format at 18°C performed at the CSIRO Collaborative Crystallization Centre , followed by in-house refinement. Diffracting crystals of CyRPA were finally obtained from hanging drops containing 9% PEG1000, 9% PEG8000, 0.25 M potassium iodide and 50 mM potassium thiocyanate. Diffracting crystals of CyRPA/Fab complex were finally obtained from hanging drops containing 11% PEG5000 monomethyl ether and 0.2M sodium acetate, pH 5.5. For data collection, crystals were transferred to cryo-protection solutions  and cryo-cooled by plunging into liquid nitrogen. X-ray diffraction data were collected on beamline MX2 at the Australian Synchrotron at a temperature of ~100 K .Diffraction data were processed and scaled with XDS . ResolutN-acetyl-α-D-neuraminic acid sodium salt (4 mU-NeuNAc). Reaction volumes (100 μl) containing 4 mU-NeuNAc (100 μM) at pH 5.0 , pH 6.5  and pH 8.0  were incubated with BSA (5 μg/ml), CyRPA (5.0 μg/ml) or Arthrobacter ureafaciens sialidase (neuraminidase)  for 4 hr at 37°C. Each reaction was quenched with glycine buffer  and fluorescence measured  on a Cary Eclipse fluorimeter (Agilent Technologies). These values, obtained in triplicate, were normalized with respect to the fluorescence measured for 10 μM 4-methylumbelliferone in glycine buffer  to determine the completeness of each reaction.Sialidase  activity was assayed using the fluorogenic substrate 4-methylumbelliferyl  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.Plasmodium falciparum protein CyRPA" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom Stephen C. Harrison (Reviewer #1), is a member of our Board of Reviewing Editors, and the evaluation has been overseen by John Kuriyan as the Senior Editor.Thank you for submitting your article "Structural basis for inhibition of erythrocyte invasion by antibodies to The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Plasmodium falciparum. They show that CyRPA interacts directly with RH5. They identify monoclonal antibodies with growth inhibitory activity and present data suggesting that one of these prevents CyRPA from interacting with Rh5. They then present the structure of CyRPA, both alone and in complex with one of these antibodies. This is an interesting piece of work that analyzes an important protein and putative vaccine candidate.Chen at all investigate the structure and inhibition of PfCyRPA, an essential protein for the invasion of erythrocytes by Major points needing attention/revision:1/2 for the outer shell are extremely low. In addition, the overall mergeRis very high, and the mergeR in the outer shell is exceptionally high. The structure of the complex is actually the better of the two; the claimed resolution is 2.26 Å, but even 2.5Å (a more conservative cutoff) is accurate enough for the conclusions the authors draw. The data processing and refinement need to be repeated using more appropriate resolution cutoffs, and the new, lower, resolution should be reported in the main text.1) The structural biology is generally convincing, but the data cut-off used is too generous. The values for I/sig(I) and CC2) In the parasite inhibition studies, a control antibody should be included for comparison. For a reader to judge how effective these CyRPA antibodies are at inhibiting invasion, it would also be valuable to include one or more other inhibitory antibodies (i.e. those that bind to PfRH5) included in this analysis so that the study would go beyond a demonstration of some inhibitory potential and allow the reader to see how inhibitory potency of this mAb compares with those of mAbs targeting other leading vaccine candidates. The field generally lacks this comparative analysis, which makes deciding which vaccine candidates to take forward challenging.3) The authors show data to suggest 8A7 directly prevents RH5 binding to CyRPA, while 7A6 does not . While t3) The authors state that they find no evidence for higher-order association from SEC of CyRPA. 4) It would be good to have a direct measurement, for example by SPR, of the CyRPA:RH5 interaction, in the presence and absence of the antibody. The relative affinities and on- and off- rates for these interactions will probe how this antibody-mediated inhibition works and show whether the kinetics of the two binding events are compatible with the disruption of a pre-formed CyRPA:CyRPA complex.5) Please provide a DNA sequence for the entire Fab , as it is important for assessing the structure.  Major points needing attention/revision:1/2 for the outer shell are extremely low. In addition, the overall Rmerge is very high, and the Rmerge in the outer shell is exceptionally high. The structure of the complex is actually the better of the two; the claimed resolution is 2.26 Å, but even 2.5Å (a more conservative cutoff) is accurate enough for the conclusions the authors draw. The data processing and refinement need to be repeated using more appropriate resolution cutoffs, and the new, lower, resolution should be reported in the main text.1) The structural biology is generally convincing, but the data cut-off used is too generous. The values for I/sig(I) and CCWe have reprocessed the data, refined the structure and modified 2) In the parasite inhibition studies, a control antibody should be included for comparison. For a reader to judge how effective these CyRPA antibodies are at inhibiting invasion, it would also be valuable to include one or more other inhibitory antibodies (i.e. those that bind to PfRH5) included in this analysis so that the study would go beyond a demonstration of some inhibitory potential and allow the reader to see how inhibitory potency of this mAb compares with those of mAbs targeting other leading vaccine candidates. The field generally lacks this comparative analysis, which makes deciding which vaccine candidates to take forward challenging.We have now included an inhibitory anti-Ripr monoclonal antibody 5G6 as a positive control and a non-inhibitory anti-Rh5 monoclonal antibody 4G4 as a negative control.3) The authors show data to suggest 8A7 directly prevents RH5 binding to CyRPA, while 7A6 does not . While tThe concentration of PfRh5 and CyRPA used in For panel 2D, we agree that anti-CyRPA antibody 7A6 that doesn’t bind to folded CyRPA is not the perfect control. Nevertheless, we have demonstrated that without a blocking antibody, Rh5 would form a complex with CyRPA.However, to address the reviewers comments we have screened a larger number of monoclonals to both PfRh5 and CyRPA. All of the CyRPA non-inhibitory monoclonals produced react to internal epitopes as is the case for 7A6 so were not useful as extra controls required by the reviewers. We have retained the experiment using 7A6 as a control as we believe it is relevant. However, we have identified a monoclonal antibody to PfRh5 (Rh5 4G4), which is non-inhibitory, and this binds to the PfRh5/CyRPA complex but does not disrupt it. This adds an additional control requested in 3) The authors state that they find no evidence for higher-order association from SEC of CyRPA. The CyRPA we used in the experiments is monomeric based on SEC analyses. In Thus, in We have altered 1B and 2C figure legends to more fully explain this.The antibody 8A7 epitope lies outside the interface of the tetrameric CyRPA in the crystallographic asymmetric unit.4) It would be good to have a direct measurement, for example by SPR, of the CyRPA:RH5 interaction, in the presence and absence of the antibody. The relative affinities and on- and off- rates for these interactions will probe how this antibody-mediated inhibition works and show whether the kinetics of the two binding events are compatible with the disruption of a pre-formed CyRPA:CyRPA complex.We agree that SPR would be very useful to have and before submitting the initial manuscript we tried to get such data with which we were confident. However, using SPR it can be very difficult to study protein-protein interaction (apart from antibody-antigen interaction). As the reviewers would know the most difficult part is regeneration of the chip to allow multiple injections to derive kinetic data. Many proteins don’t survive the regeneration conditions and this was our major problem. Furthermore, it is very difficult to control how the protein is coupled to a chip and to find a valid control. As said above we attempted SPR to obtain the data requested by the reviewer but were unable to generate reliable data that we were comfortable in publishing.Despite this we believe that our SEC data shows that PfR5 and CyRPA form a stable complex. As stated above the CyRPA we have generated appears to be a monomer.5) Please provide a DNA sequence for the entire Fab , as it is important for assessing the structure. We have included a supplemental figure for the sequence of the entire Fab fragment.


CCCTC‐binding factor (CTCF) is a highly conserved and ubiquitously expressed C2H2‐type zinc finger protein which is considered to be involved in epigenetic memory through regulation of higher order chromatin architecture. However, the regulatory mechanism of CTCF in mitosis is still unclear. Here we found that the DNA‐binding activity of CTCF is regulated in a phosphorylation‐dependent manner during mitosis. The linker domains of the CTCF zinc finger domain were found to be phosphorylated during mitosis. The phosphorylation of linker domains impaired the DNA‐binding activity in vitro. Mutation analyses showed that amino acid residues  located in the linker domains were phosphorylated during mitosis. Based on these results, we propose that the mitotic phosphorylation of the linker domains of CTCF is important for the dissociation of CTCF from mitotic chromatin.During mitosis, higher order chromatin structures are disrupted and chromosomes are condensed to achieve accurate chromosome segregation.  ChIPchromatin immunoprecipitationCTCFCCCTC‐binding factorZFzinc fingerCis‐acting regulatory DNA elements such as insulators and enhancers are involved in the temporal and cell type‐specific control of gene expression through the formation of higher order chromatin structure. Higher order chromatin structure is tightly regulated throughout the cell cycle to achieve proper and dynamic chromosome processes. CCCTC‐binding factor (CTCF) is a transcription factor containing 11 highly conserved zinc finger motifs , cells were released into a fresh culture medium without an excess amount of thymidine for 10 h, and then synchronized again at G1/S boundary in growth medium with 2.5 mm thymidine for 14 h. At 6 h post release from thymidine treatment, cells were treated with 165 nm nocodazole (Sigma‐Aldrich Co. LLC.) for 6 h. Mitotic cells were collected by gentle shaking of cell culture dishes. Transient DNA transfection assays were performed using Gene Pulser Xcell System  according to the manufacturer's protocol.HeLa S3 cells and MCF‐7 cells were maintained at 37 °C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. Cells were synchronized at mitotic phase by two cycles of “excess thymidine blockage followed by nocodazole arrest”. Briefly, at 12 h post treatment of 2.5 mTo make 3 × FLAG‐tagged CTCF WT expression vector, CTCF cDNA fragment from pBS‐FLAG‐CTCF A rabbit polyclonal anti‐CTCF antibody was prepared as previously described m HEPES‐NaOH, pH 8.0, 10 mm KCl, 1.5 mm MgCl2, 0.1% Triton X‐100, 5 mm Na3VO4, 10 mm NaF, 25 mm glycerol 2‐phosphate) on ice for 5 min. Supernatant and pellet fractions were obtained by centrifugation at 2300 g for 5 min.Cells were washed with cold PBS(−), and kept on ice for 5 min in a cold hypotonic buffer . After centrifugation at 2300 g for 5 min, pellet fractions were incubated at 25 °C for 10 min in CSK buffer containing 0.5% formaldehyde, and then incubated with 25 °C for 5 min in 0.125 m glycine/PBS. Immunoprecipitation assays were carried out according to the manufacturer's protocol . The quantitative PCR (qPCR) was performed with primers 5′‐GGTCCACGGGCCGCCCTGCCAG‐3′ and 5′‐CGCAGCTCCGGAAGCCGAGAGC‐3′ corresponding to a part of rRNA gene between nucleotide positions −961 and −851, where +1 is set to be the transcription start site Cells were washed with cold PBS(−), and kept on ice for 5 min in a cold CSK buffer . Nocodazole‐arrested MCF‐7 cells were incubated on ice for 5 min in the hypotonic buffer. CTCF was immunoprecipitated from the lysates using the anti‐CTCF antibody. After washing with a buffer containing 10 mm Tris/HCl, pH 7.9, 500 mm NaCl, 1 mm MgCl2, 0.1% Triton X‐100, 1 mm Na3VO4, 1 mm NaF, 5 mm glycerol 2‐phosphate, immunoprecipitated proteins were incubated with or without lambda protein phosphatase  at 30 °C for 2 h, and then incubated in a DNA‐binding buffer  with 0.15 ng of the rRNA gene upstream region fragment and 50 ng of poly (dIdC). The rRNA gene upstream region fragment used here is amplified from genomic DNA and purified from agarose gel. The exact sequence of the fragment corresponding to the region between nucleotide positions −961 and −851, where +1 is set to be the transcription start site imagej software  from western blotting results.Asynchronously growing MCF‐7 cells were incubated on ice for 5 min in the hypotonic buffer. Supernatant and pellet fractions were separated by centrifugation at 2300 rRNA gene promoter rRNA gene locus during mitosis by chromatin immunoprecipitation (ChIP) assays. The amount of CTCF on the rRNA gene locus in mitotic cells was 80% less than that in asynchronous cells (Fig. rRNA gene was restored (Fig. It is reported that most of the C2H2 zinc finger family proteins are excluded from mitotic chromosomes c‐mycIt has been reported that a variety of DNA‐binding factors including transcription factors are phosphorylated and released from highly condensed chromosomes during mitosis It has been reported that serine residues at amino acid positions 604, 609, 610, and 612 of CTCF are phosphorylated by protein kinase CK2 in vitro DNA‐binding assays. The purified CTCF, bound to protein A agarose beads, was treated with or without lambda protein phosphatase, and incubated with a fragment of the rRNA gene that contains the CTCF‐binding site. Quantification of fragments that interacted with CTCF was accomplished by qPCR and normalized against the protein amount of CTCF that was eluted from protein A agarose beads and quantified by western blotting. The DNA‐binding activity of mitotic CTCF was 80% lower than asynchronous CTCF (Fig. rRNA gene locus bound to 3 × FLAG‐CTCF 8D was reduced by 30% compared to wild‐type (Fig. To determine whether the mitotic phosphorylation of linker domains affects the DNA‐binding activity, CTCF was purified using anti‐CTCF antibody from either asynchronous or mitotic MCF‐7 cell extracts and subjected to TCF Fig. A. HoweveTCF Fig. A. These TCF Fig. B. To anaype Fig. C. These It has been reported that the phosphorylation of linker domains reduces the DNA‐binding activity of C2H2 zinc finger proteins It has been proposed that mitotic chromosomes exist in a highly condensed state to ensure stable and integral chromosome segregation rRNA upstream regions. These results suggest that the DNA‐binding activity of CTCF could be regulated through the phosphorylation of linker domains during mitosis.Here we have shown that CTCF dissociates from mitotic chromosomes. Mutation analyses indicated that CTCF is phosphorylated in mitosis at Thr289, Thr317, Thr346, Thr374, Ser402, Ser461, and Thr518, all of which are located in linker domains. The mitotic phosphorylation of CTCF resulted in the reduction of the DNA‐binding activity to a CTCF‐binding site on TS conceived the research strategies, performed experiments, analyzed the data, and wrote the manucsript. KS designed the experiments and analyzed the data. KK and AK designed the experiments, analyzed the data, and wrote the manuscript. KN supervised the research and wrote the manuscript.


The atypical kinase Rio1 is widespread in many organisms, ranging from Archaebacteria to humans, and is an essential factor in ribosome biogenesis. Little is known about the protein substrates of the enzyme and small-molecule inhibitors of the kinase. Protein kinase CK2 was the first interaction partner of Rio1, identified in yeast cells. The enzyme from various sources undergoes CK2-mediated phosphorylation at several sites and this modification regulates the activity of Rio1. The aim of this review is to present studies of the relationship between the two different kinases, with respect to CK2-mediated phosphorylation of Rio1, regulation of Rio1 activity, and similar susceptibility of the kinases to benzimidazole inhibitors. Protein kinases play important roles in key cellular processes, including the cell cycle, metabolism, and cell death ,2. The pThe objective of the review article is to present a representative of the RIO family, namely the atypical protein kinase Rio1. The special intention is to emphasize the relationship of Rio1 from humans and yeast, with probably the most pleiotropic cellular kinase—CK2. Although the interaction between these two remarkably different kinases  has not RIO kinases comprise an atypical kinase family composed of four subfamilies: Rio1, Rio2, RioK3, and RioB. The members of the RIO family are evolutionarily conserved and are present in all three domains of life. RIO kinases exhibit a trimmed version of the domain present in the canonical eukaryotic protein kinases, which lack the activation loop and the substrate recognition region ,17,18. TThe N-terminal lobe is connected via a “hinge region” to a C-terminal lobe, containing three α-helices  and a β-hairpin β6-7) . In cont . In conThe enzyme is involved in processes critical for cellular proliferation, including ribosome biogenesis, cell cycle progression, and chromosome maintenance ,20,21. BWhile the cytoplasmic functions of Rio1 are well characterized, little is known about its role in the nucleus, where it is also present. Recent studies have revealed that, Rio1 downregulates RNA polymerase I (Pol I) during anaphase by targeting its subunits Rpa43, thereby causing the polymerase to dissociate from rDNA; thus, Rio1 integrates rDNA replication and segregation with ribosome biogenesis .in vitro, namely, myelin basic protein, casein, histones H1 and H2A, enolase, Tomato mosaic virus MP, and the recently discovered subunit of RNA Pol I—Rpa43 [Interestingly, the protein kinase Rio1 is characterized as an essential enzyme involved in cell viability; on the one hand; on the other hand only one physiological protein substrate (Rpa43) for this kinase is known. There are several proteins that undergo Rio1-mediated phosphorylation  I—Rpa43 ,10,13,25 I—Rpa43 ,8. It hawww.phosphosite.org). All the sites mentioned have been revealed by High Throughput Screening, but not confirmed by site-specific methods. Among all these phosphosites, there are three  located within two sequences that fulfil the minimal consensus for phosphorylation by protein kinase CK2 [S21S22DSENRDL [There are 18 phosphorylated amino acids within the hRio1 sequence listed at PhosphoSitePlus  were identified as CK2 phosphorylation sites by means of site-directed mutagenesis. Consecutive mutation of the listed residues resulted in a decreased phosphorylation level, up to total abrogation of the signal in the case of mutation of all six serines. A Rio1 mutant mimicking the permanently phosphorylated enzyme, with the serines replaced with aspartic acids, showed approximately two fold higher phosphorylation activity toward histone H2B in comparison with the wild-type kinase and a Rio1 mutant lacking CK2-phosphorylated sites (S > A). In order to examine the possible biological importance of the in vitro studies, yeast cells harbouring a Rio1 mutant lacking serines modified by CK2 or a Rio1 mutant mimicking the state of permanent CK2-mediated phosphorylation were employed. The former yeast cells showed a significantly reduced growth rate, while the latter cells behaved indistinguishably from the wild-type yeast. The results show that phosphorylation of CK2-modified serines is essential for full activity of the kinase Rio1, and lack of the phosphorylation is disadvantageous for cell proliferation. Further, the authors revealed that the yeast cells harbouring the Rio1 mutant, which are not phosphorylated by CK2, had difficulties entering the S phase. Moreover, it has been shown that the S > A mutant shows low susceptibility to proteolytic degradation in contrast to the wild-type Rio1 and the S > D mutant .Nicotiana tabacum inhibited the interaction between Rio1 and the Movement Protein from Tomato Mosaic Virus. The authors suggest that phosphorylation occurs in the region of protein-protein interaction, and the process disrupts protein binding through changes in the protein conformation of Rio1 [In other studies, recombinant tobacco CK2-mediated phosphorylation of Rio1 from  of Rio1 .Although the possible role of Rio1 in pathogenesis is not well established in comparison to many other kinases, it has been reported that the enzyme is upregulated in colon cancer, and there is a direct link between deregulation of ribosome biogenesis and tumor development . In othe50 of 48 µM (AG490), at the ATP concentration of 1 µM which is optimal for Rio1, is rather low.Currently there are not selective compounds targeting Rio1. The first small-molecule inhibitor of protein kinase Rio1 discovered is an antibiotic toyocamycin (TOYO). It is capable of inhibiting both ribosome biogenesis and kinase activity of Rio1 . The antDerivatives of benzimidazoles are widely reported inhibitors of protein kinase CK2 from different sources, which show pro-apoptotic properties in tests on cancer cell lines and mouse xenografts ,33,34,35Our recent studies have revealed that Rio1 is inhibited in vitro by selected benzimidazoles with potency similar to that of CK2 . The comInterestingly, Rio1 has not been tested before with other kinases in studies on the selectivity of benzimidazole inhibitors of CK2. Our data have shown that the most potent compound, i.e., TIBI, can act as a strict ATP-competitive inhibitor of the atypical kinase. Since the first inhibitor of Rio1 discovered, i.e., toyocamycin was reported to stabilize the less catalytically active oligomer and to enhance the thermostability of the kinase, we attempted to verify if these phenomena are general properties of the ATP-competitive inhibitors of Rio1. The results obtained have shown that TIBI does not influence the ternary structure of Rio1 but enhances the thermostability of the kinase. Molecular docking calculations have revealed that TIBI binds to the ATP-binding pockets of both kinases in a similar manner .Although the nanomolar activity of some benzimidazoles against Rio1 could be a promising starting point in designing novel inhibitors of the kinase, the fact that these compounds target also many other kinases creates a serious barrier to reaching reasonable selectivity. On the other hand, taking into consideration the activity of toyocamycin against Rio1 and its selectivity with respect to CK2, the derivatization of the antibiotic appears to be a more attractive approach in designing potent and selective Rio1 inhibitors.Summing up, there are some relationships between Rio1 and CK2. The enzymes can interact with one another. Rio1 undergoes CK2-mediated phosphorylation and this modification can regulate Rio1 activity. Although the articles reviewed here clearly show the importance of CK2 activity in Rio1 regulation, further studies should be carried out, mainly on human cells, in order to elucidate this interaction. However, the most intriguing aspect of their relationship is the shared susceptibility to benzimidazole inhibitors. This issue should be taken into consideration when new benzimidazole-based biologically active chemicals are designed. Considering the limited selectivity of benzimidazoles, the chemicals are not promising candidates for selective Rio1 inhibitors. However, benzimidazoles targeting a variety of kinase overexpressed in some pathological states, are widely used as anti-kinases agents. The novel anti-Rio1 activity of benzimidazoles discovered recently should be taken into consideration whenever new compounds are tested against a panel of protein kinases, and in such a case the atypical protein kinase Rio1 cannot be disregarded.