Neuronal precursor cell expressed developmentally downregulated 4-2 (NEDD4-2) is an E3 ubiquitin ligase to regulate ion transport by controlling cellular trafficking/endocytosis and lysosomal degradation of ion channels and transporters. Thus, NEDD4-2 is relevant to neuronal excitability and epileptic encephalopathies in human patients. However, the regulatory molecules for NEDD4-2 dephosphorylation have been still elusive. Here, we demonstrate that pyridoxal-5′-phosphate phosphatase/chronophin (PLPP/CIN) specifically dephosphorylated NEDD4-2 serine (S) 448 site. PLPP/CIN deletion inhibited NEDD4-2 ubiquitination, and diminished the responsiveness of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (AMPAR) by facilitating NEDD4-2-mediated ubiquitination of GluA1 subunit under physiological condition. PLPP/CIN overexpression reversed these effects. These PLPP/CIN-mediated processes were required for the increased seizure severity and its progression in response to kainic acid (KA). Therefore, we suggest the novel function of PLPP/CIN as a NEDD4-2 phosphatase, which may be a potential therapeutic target for NEDD4-2-associated diseases as well as various neurological and psychiatric disorders, including epilepsy. However, the cellular and molecular mechanisms in epilepsy remain unclear.Epilepsy is one of the common neurological disorders, suffering about 1% of all people, which is characterized by the periodic occurrence of seizures exhibiting abnormal synchronized neuronal discharges. Unprovoked recurrent seizures contribute to a cyclical or progressive process of worsening epilepsy and neurological deficits including learning disabilities and memory problems. The pathophysiology underlying seizure susceptibility is relevant to channelopathy, aberrant synaptic organization, impaired glial function, inflammation, and neuronal loss11.Ubiquitination is a rapid, local, and reversible posttranslational modification that involves the covalent conjugation of ubiquitin to target proteins, which assigns misfolded cytosolic proteins for degradation by the 26S proteasome and regulates physiological processes. Ubiquitination is involved in modulation of synaptic function via nonproteolytic processes such as endocytosis, protein localization/targeting, complex assembly and regulation of the duration, and intensity of signaling by effector molecules. Generally, ubiquitin is bound to lysine residues on target proteins by a cascade of reactions carried out sequentially by the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase enzyme (E3). In the final step, an E3 ubiquitin ligase transfers the ubiquitin to recognition motifs in the target proteins and thus confers target protein specificity17. Therefore, it is required for optimal regulation of NEDD4-2-mediated channel degradation/internalization to maintain neuronal excitability. Indeed, NEDD4-2 mutations are relevant to epileptic encephalopathies in human patients20. In addition, Nedd4-2andi mice, in which the long-form (isoform 1) of NEDD4-2 is selectively deleted due to a spontaneous mutation in exon-2, show the elevated kainic acid (KA)-induced seizure susceptibility. The fidelity and activation of NEDD4-2 activity is regulated by phosphorylations that which disrupt target channels-NEDD4-2 interactions22. Oppositely, NEDD4-2 phosphorylations are required for a substrate ubiquitination24 and maintenance of its stability12. Although the phosphorylation of human NEDD4-2 on serine (S) 342  and S448  residues by various kinases including serum glucocorticoid kinase (SGK1)27, furthermore, little is known yet to explain the regulatory molecules for NEDD4-2 dephosphorylation.Neuronal precursor cell expressed developmentally downregulated 4-2 (NEDD4-2) is an E3 ubiquitin ligase to control cellular trafficking/endocytosis and lysosomal degradation of ion channels and transporters28. Recently, we found that PLPP/CIN dephosphorylates calsenilin (CSEN) and reversely regulates CSEN bindings to Kv4.2 (an A-type K+ channel) and N-methyl-D-aspartate receptor independent of cofilin-mediated F-actin dynamics. Furthermore, PLPP/CIN deletion exhibits the lower intensity (severity)29, duration, and progression of seizures, but the shorter latency of seizure onset in response to KA. PLPP/CIN overexpression reverses these phenomena30. These novel properties of PLPP/CIN provide the possibility that interaction of PLPP/CIN with other biological molecules may modulate neuronal hyperexcitability independent of F-actin depolymerization.Pyridoxal-5′-phosphate phosphatase/chronophin (PLPP/CIN) dephosphorylates and activates cofilin, which depolymerizes F-actin+ channels KCNQ2/3/5. We further describe that NEDD4-2 knockdown increased seizure severity and facilitated its progression in response to KA, like PLPP/CIN overexpression. These findings indicate the novel function of PLPP/CIN as a NEDD4-2 phosphatase, which may regulate neuronal excitability by an inhibitory mechanism for E3 ubiquitin ligase activity of NEDD4-2. Therefore, we suggest that PLPP/CIN may be a potential therapeutic target for epileptogenesis and NEDD4-2-associated diseases.Here, we demonstrate that PLPP/CIN bound to NEDD4-2 and dephosphorylated its S448 site, which accelerated NEDD4-2 ubiquitination, and subsequently enhanced the responsiveness of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor by inhibiting NEDD4-2-mediated ubiquitination of GluA1 subunit, but not voltage-gated KPLPP/CIN−/−) mice showed the increases in NEDD4-2 protein and S448 phosphorylation levels to 1.8-fold and 3-fold of WT mice levels, respectively  was ~0.6-fold of WT mice level , and seizure duration in response to KA , which is required for stabilizing the resting membrane potential and limiting neuronal excitability, by ubiquitinating KCNQ2/3/5 channels34. Indeed, mutations in KCNQ are associated with various epilepsy phenotypes including benign familial neonatal seizures37. Therefore, it is likely that PLPP/CIN-mediated NEDD4-2 ubiquitination would affect KA-induced seizure activity by altering surface KCNQ2/3/5 expressions. To elucidate this hypothesis, we explored the membrane expression levels of KCNQ2/3/5 in PLPP/CIN−/− mice. However, there was no differences in total and membrane KCNQ2/3/5 expressions between WT and PLPP/CIN−/− mice, which were unaffected by KA injection , which is mediated by AMPAR28, we accessed whether PLPP/CIN deletion affects NEDD4-2-mediated GluA1 ubiquitination. Consistent with our previous study28, there was no difference in total GluA1 protein level between WT and PLPP/CIN−/− mice  into the dentate gyrus29. As compared to WT mice, the efficacy of AMPA-mediated neuronal discharge was lower in PLPP/CIN−/− mice  mice. Consistent with our previous study30, PLPP/CINTg mice showed the increases in the seizure latency and seizure intensity (severity) in response to KA  phosphorylation levels without altering SGK1 activity 23, and cardiac voltage-gated Na+ channel40. However, phosphorylations cannot completely suppress NEDD4-2 function41. Indeed, NEDD4-2 phosphorylation does not affect the ubiquitination of neuronal voltage-gated Na+ channels19. Furthermore, NEDD4-2 phosphorylation is required for a ENaC ubiquitination23. In the present study, we found that PLPP/CIN bound to NEDD4-2 and reduced S448 phosphorylation without changing NEDD4-2 S342 phosphorylation, and subsequently diminished NEDD4-2-mediated GluA1 ubiquitination. Since PLPP/CIN deletion and overexpression cannot influence the expression levels and activities of protein phosphatase 1A, protein phosphatase 2A, and protein phosphatase 2B under physiological condition and following KA injection30, our findings suggest that PLPP/CIN may specifically dephosphorylate S448 residue on NEDD4-2, and inhibit NEDD4-2 E3 ubiquitin ligase activity by reducing NEDD4-2 protein level.The novel findings in the present study are that PLPP/CIN dephosphorylates NEDD4-2 S448 site under physiological and postseizure conditions. NEDD4-2 phosphorylation increases surface expressions of KCNQ25. In the present study, PLPP/CIN deletion increased NEDD4-2 protein level with enhancing NEDD4-2 S448 phosphorylation and reducing NEDD4-2 ubiquitination under physiological condition and after KA injection, while PLPP/CIN overexpression showed the reverse effects. With respect to ubiquitination of NEDD4-233, these findings suggest that PLPP/CIN-mediated NEDD4-2 S448 dephosphorylation may be one of inhibitory mechanisms for regulation of NEDD4-2 activity via its ubiquitination. In the present study, furthermore, PLPP/CIN deletion reduced the phosphorylation ratio  of S342, but elevated that of S448, concomitant with the increase in total NEDD4-2 protein level, which were reversed by PLPP/CIN overexpression. Since NEDD4-2 phosphorylation inhibits ubiquitinations of various ion channel and maintains its protein level40, it is plausible that PLPP/CIN would inhibit NEDD4-2 E3 ubiquitin ligase activity by reducing target channel-NEDD4-2 interaction and its protein level by increasing S342 and diminishing S448 phosphorylation ratio, respectively. However, the present data could not provide the evidence for the role of S342 phosphorylation ratio in NEDD4-2-substrate interactions. Furthermore, the present study could not verify whether NEDD4-2 ubiquitin modification results from self-ubiquitination or the activations of other unknown E3 ligases, although NEDD4-2 is auto-ubiquitinated like other E3 ligases33. Thus, it would be needed to elucidate the role of the relative NEDD4-2 phosphorylation ratio in its activity, and the specific E3 ligase for NEDD4-2 ubiquitination in future studies.Aforementioned, S448 site plays a role in NEDD4-2 protein stability independent of 14-3-3 bindings, since inhibition of NEDD4-2 S448 phosphorylation reduces NEDD4-2 protein level43. Thus, perturbation of the homeostatic scaling following stimulation may trigger a prolonged neuronal hyperexcitability inducing seizure activity24. In the present study, PLPP/CIN deletion interrupted seizure progression induced by KA, which was reversed by NEDD4-2 knockdown. Consistent with the present data, Nedd4-2andi mice show the elevated KA-induced seizure susceptibility14. The present study also reveals that the enhancements of PLPP/CIN-NEDD4-2 bindings and NEDD4-2 ubiquitination in PLPP/CINTg mice increased seizure intensity and its progression in response to KA. Thus, our findings indicate that PLPP/CIN-mediated NEDD4-2 ubiquitination may enhance seizure activity and lead to seizure progression.NEDD4-2-mediated ubiquitination is one of the underlying machineries for synaptic homeostatic scaling, which is a feedback mechanism for maintenance of a physiological rage of neuronal excitabilityNedd4-2andi mice is recovered by the genetically reducing GluA1 level14. Furthermore, Fmr1−/− mice (the fragile X syndrome mouse model) show neuronal hyperexcitability, because loss-of-function dephosphorylation of NEDD4-2 impairs NEDD4-2-GluA1 interaction and GluA1 ubiquitination after chronic activity stimulation, but not under physiological condition24. Therefore, it is likely that NEDD4-2 may play an important role in downregulation of synaptic homeostatic scaling via GluA1 ubiquitination14. In the present study, PLPP/CIN regulated membrane GluA1 expression and its ubiquitination under physiological condition. Furthermore, PLPP/CIN increased the seizure intensity due to the reduced GluA1 ubiquitination following KA injection. Since PLPP/CIN enhances the synaptic strength during high-frequency stimulation28, our findings suggest a novel mechanism by which PLPP/CIN inhibits synaptic downscaling by dephosphorylating NEDD4-2 S448 site and disrupting the subsequent GluA1 ubiquitination during neuronal hyperexcitability. Conversely, it is plausible that GluA1-NEDD4-2 bindings would be affected by GluA1 phosphorylation level, since the Nav1.6 S553 phosphorylation by p38 mitogen-activated protein kinase is required for its binding to NEDD4-244. Under physiological conditions, however, PLPP/CIN deletion/overexpression cannot affect GluA1 S831 and S845 phosphorylation levels28. Therefore, our findings also indicate that NEDD4-2-mediated GluA1 ubiquitination may be independent of GluA1 phosphorylation.Recently, it has been reported that the higher seizure susceptibility due to insufficient function of NEDD4-2 in Fmr1−/− mice24, the activity-dependent dephosphorylation of NEDD4-2 is caused by p53-medaited AKT destabilization, which is an upstream pathway of SGK145. In the present study, KA reduced SGK1 activity and S342 and S448 phosphorylations of NEDD4-2. Thus, it is likely that activity-dependent SGK1 inhibition would also be involved in NEDD4-2 dephosphorylation. However, PLPP/CIN expression level influenced only NEDD4-2 S448 phosphorylation, but not NEDD4-2 S342 and SGK1 phosphorylation, under physiological and postseizure conditions. Therefore, our findings suggest that PLPP/CIN may fine-tune NEDD4-2-GluA1 interaction and GluA1 ubiquitination by S448 dephosphorylation under basal and pathological conditions independent of SGK1 activity.In the case of 47. NEDD4-2 phosphorylation at S448 site is essential for the SGK1-dependent regulation of KCNQ2/3 and KCNQ3/5 channels in Xenopus oocytes39. Considering E3 ubiquitin ligase activity of NEDD4-2 for KCNQ34, it is likely that PLPP/CIN-mediated NEDD4-2 ubiquitination would affect the surface KCNQ2/3/5 expressions. However, PLPP/CIN deletion/overexpression and NEDD4-2 knockdown did not show any significant effect on total or surface KCNQ2/3/5 expression, suggesting the dominant role of NEDD4-2 in GluA1 ubiquitination. These data demonstrate that PLPP/CIN may not influence the general ubiquitination process mediated by NEDD4-2. Since NEDD4-2 ubiquitinates multiple membrane receptors, including voltage-gated Na+ channel16, neurotrophin receptors17, and glutamate transporters15, however, the possibility is not excluded that changes in ubiquitination of other substrates could contribute to seizure activity when PLPP/CIN are deleted or overexpressed. Thus, further studies are needed to elucidate whether and how PLPP/CIN mediates ubiquitination status of other NEDD4-2 substrates, which contributes to seizure generation and/or its progression.Impaired KCNQ regulation causes spontaneous seizures due to reduction in M-current that exerts a stabilizing effect on the resting membrane potential and attenuates neuronal excitability14 have revealed that NEDD4-2 mutation increases seizure susceptibility in response to KA. Interestingly, the same group has also reported that murine double minute-2 triggers the degradation of p53 and subsequent induction of NEDD4-2 as an adaptive response in neuronal culture upon chronic elevation of neuronal activity by picrotoxin, a GABAA receptor antagonist38. In the present study, however, NEDD4-2 expression level was reduced in the hippocampus of WT mice 2 h after KA injection. Similar to the present data, pilocarpine (a muscarinic acetylcholine receptor agonist)-induced acute seizure activity rapidly degrades NEDD4-2 in the rat hippocampus 2 h after injection48. These discrepancies would result from the differences in methods , seizure severity in response to convulsants (picrotoxin vs. pilocarpine or KA), or dose of KA (60 vs. 25 mg/kg). However, the data from these previous reports and the present study identically suggest that the reduced NEDD4-2 expression may increase seizure severity and its duration. Indeed, chronic epilepsy rat shows downregulation of NEDD4-2 expression48, and NEDD4-2 knockdown increased seizure severity and its duration in PLPP/CIN−/− mice in the present study. Furthermore, the present data demonstrate that PLPP/CIN activity in PLPP/CINTg mice was higher than that in WT mice under physiological condition. PLPP/CIN overexpression also diminished NEDD4-2 protein level with enhancing NEDD4-2 S448 dephosphorylation and its ubiquitination under physiological condition. In addition, KA enhanced NEDD4-2 S448 dephosphorylation in WT and PLPP/CINTg mice without altering PLPP/CIN activity, indicating that seizure activity may increase NEDD4-2 dephosphorylation via promoting physical interaction between PLPP/CIN and NEDD4-2. Therefore, our findings suggest that PLPP/CIN-mediated NEDD4-2 dephosphorylation may play an important role in the seizure intensity and its progression, regardless of the presence of compensatory upregulation of NEDD4-2 expression in response to seizures48.On the other hand, Zhu et al.In conclusion, we describe a new paradigm for the regulation of NEDD4-2 activity by PLPP/CIN-mediated S448 dephosphorylation Fig. . To our PLPP/CIN−/− (129/SvEv-C57BL/6J background) and PLPP/CINTg (C57BL/6J background) mice were used in the present study. Each background WT mice were used as control animals, respectively. Animals were provided with a commercial diet and water ad libitum under controlled temperature, humidity, and lighting conditions . All experimental protocols were approved by the Animal Care and Use Committee of Hallym University. All reagents were obtained from Sigma-Aldrich , except as noted.30. NEDD4-2 (10 ng/μl) was phosphorylated by incubation with 200 U/μl SGK1  and 100 μM ATP in the kinase assay buffer I  at 30 °C for 1 h. Thereafter, the sample was portioned the same volume, added PLPP/CIN (10 ng/μl) or 50 mM Tris buffer (control), and incubated at 30 °C for 1 h. Crude extracts obtained from the same PLPP/CIN−/− mice were used the same method omitting SGK1 and NEDD4-2. Thereafter, the samples were used coprecipitation and western blot analysis (see below).Modified in vitro PLPP/CIN phosphatase assay using full-length recombinant human NEDD4-2  and human PLPP/CIN proteins  was performed as described previously2O:O2). Surgery for a brain infusion kit and an electrode implantation was performed according to our previous study30. A brain kit 3  was inserted into the lateral cerebral ventricle (2.0 mm depth from bregma), and connected with a 1007D Alzet osmotic pump  containing control siRNA (20 μM) or NEDD4-2 siRNA (20 μM), respectively. siRNA sequence targeting NEDD4-2 corresponding to coding region (5′→3′) are sense: AAACUCUCUGGAGUACGGAACAGCCUU, and antisense: GGCUGUUCCGUACUCCAGAGAGUUUUU, or sense: UUCAGAUCCACUUGGUAUGUCUGCCUU, and antisense: GGCAGACAUACCAAGUGGAUCUGAAUU. Non-silencing RNA (5′-GGCGCGCTTTGTAGGATTCGA-3′) was used as the control siRNA . Osmotic pump was implanted subcutaneously in the midscapular region of the back. Monopolar electrode  or a guide-electrode-combo  was also implanted into the left dorsal hippocampus .Under anesthesia with isoflurane  was administered 2 h after KA injection and repeated, as needed. After recording, animals were quickly decapitated, and their hippocampi were dissected out in the presence of cooled artificial cerebrospinal fluid  and stored −80 °C until preparation for biochemical experiments30.After baseline recording for at least 30 min, animals were given KA . Control animals received an equal volume of normal saline instead of KA. EEG signals were recorded with a DAM 80 differential amplifier  and the data were digitized (1000 Hz) and analyzed using LabChart Pro v7 software . Latency of seizure onset was defined as the time point showing more than 3 s and consisting of a rhythmic discharge between 4 and 10 Hz with amplitude of at least two times higher than the baseline EEG29. EEG signals were digitized and analyzed using LabChart Pro v7 (AD Instruments) by the same methods aforementioned.After baseline recording for at least 30 min, animals implanted with a guide-electrode-combo  were directly infused AMPA (20 μM) over a 1-min period using a microinjection pump  into the hippocampus with an internal infusion cannula ®, Roche Applied Science, USA) and 1 mM sodium orthovanadate. Protein concentrations were calibrated by the BCA protein assay  and equal amounts of total proteins were incubated with NEDD4-2, Ubiquitin, or GluA1 antibody  containing protease inhibitor cocktail , phosphatase inhibitor cocktail , according to the manufacturer’s instructions.Western blotting was performed according to standard procedures. The list of primary antibody used in the present study is Supplementary Table 50. All images were obtained using an AxioImage M2 microscope and AxioVision Rel. 4.8 software. Areas of interest (1 × 105 μm2) were selected in the captured images of the CA3 region of the hippocampus proper , and the number of FJB-positive neurons was counted50.One day after KA injection, animals were perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) under urethane anesthesia . Brains were postfixed in the same fixative overnight and then cryoprotected and sectioned at 30 μm with a cryostat. Thereafter, tissues were used for a conventional FJB staining according to previous studiesW-test, Student’s t test or ANOVA were used to analyze statistical significance. Bonferroni’s test was applied for post hoc comparisons. A p-value below 0.05 was considered statistically significant. Quantitative data were expressed as mean ± standard error of the mean.After evaluating the values on normality using Shapiro–Wilk Supplemental material


It is a potential drug target in the treatment of type II diabetes. The protein is also associated with a rare inherited metabolic disease and some cancer cells lack FBPase activity which promotes glycolysis facilitating the Warburg effect. Thus, there is interest in both inhibiting the enzyme (for diabetes treatment) and restoring its activity (in relevant cancers). The mammalian enzyme is tetrameric, competitively inhibited by Fructose 2,6-bisphosphate and negatively allosterically regulated by AMP. This allosteric regulation requires information transmission between the AMP binding site and the active site of the enzyme. A recent paper by Topaz et al. (Bioscience Reports (2019) 39, pii:BSR20180960) has added additional detail to our understanding of this information transmission process. Two residues in the AMP binding site (Lys112 and Tyr113) were shown to be involved in initiating the message between the two sites. This tyrosine residue has recently be shown to be important with protein’s interaction with the antidiabetic drug metformin. A variant designed to increase metal ion affinity (M248D) resulted in a five-fold increase in enzymatic activity. Interestingly alterations of two residues at the subunit interfaces (Tyr164 and Met177) resulted in increased responsiveness to AMP. Overall, these findings may have implications in the design of novel FBPase inhibitors or activators.Fructose 1,6- This rehosphate . In conthosphate . The con purpose .bisphosphate inhibition is competitive, with the compound binding to the active site and sterically hindering access by the substrate, fructose 1,6-bisphosphate [FBPase is a homotetramer in most species studied to date, with yeasts being a notable exception ,10. Likehosphate . In conthosphate . These thosphate ,12. It ahosphate .FBPase inhibition has been suggested as a potential therapy for type II diabetes ,14. In tFBPase is associated with a rare, autosomal recessive inherited metabolic disease OMIM #229700). The incidence is estimated to be between 1/900000 and 1/350000 in European populations . Disease700. The In clear cell renal cell carcinoma , cells lack FBPase activity, due to a chromosomal deletion . ComplemThe existence of allostery in FBPase infers the presence of information transmission pathways within the protein. Typically, allosteric changes are transmitted through proteins by conformational changes, alterations in mobility or a combination of the two . CrystalRecent work by Topaz et al. , publishbisphosphate. The positively co-operative response to AMP was abolished by these changes. These variants also had increased catalytic efficiency (as measured by the specificity constant kcat/Km). A Y113F variant (Y114F in the numbering scheme used in the paper reporting this variant) has been recently reported to have greatly reduced sensitivity to AMP, but also to fructose 2,6-bisphosphate, suggesting that this is a critical region in the AMP binding site [As expected, alteration of the two lysine residues in the AMP binding site resulted in a lower responsiveness to regulation by this compound, but did not affect regulation by fructose 2,6-ing site . It appebisphosphate. Overall, they are less enzymatically active, but retain positive co-operativity towards AMP. The two variants in the allosteric communication pathway had little effect on AMP responsiveness or catalytic efficiency and a small effect on fructose 2,6-bisphosphate responsiveness. These are an interesting set of results since these changes might have been expected to disrupt communication. That they (largely) did not do so suggests the possibility of alternative information transmission pathways. Alternatively, it is possible that the alterations to these residues are not sufficient to fully disrupt the transmission of information. This may be particularly the case if protein mobility, as well as conformational changes, is important in mediating the allosteric effects. Such alterations are often critical in the transmission of information within proteins [Interestingly, the two variants located at the subunit interface resulted in a slight increase in responsiveness to both AMP and fructose 2,6-proteins ,46,47. Fbisphosphate. As predicted, its affinity for magnesium is increased . Thus, the authors’ prediction that engineering FBPase to bind more tightly to the metal ion will result in increased activity was vindicated. Increases in catalytic efficiency and turnover are relatively rare outcomes of enzyme engineering experiments. In general, most changes reduce substrate affinities, turnover and efficiency [While all the alterations of residues to alanine were designed to abolish interactions within the protein or between the protein and its ligands, the M248D variant was intended to increase the affinity for divalent metal ions. This variant has an impressive (greater than five-fold) increase in catalytic efficiency combined with a substantially reduced responsiveness to fructose 2,6-While these experiments shed new light on the molecular mechanisms of information transmission in mammalian FBPase, the story is not complete. A map of the changes resulting from AMP binding and how these affect catalysis is not yet available. Nevertheless, each new set of data on FBPase variants which affect the process increases the knowledge base which can be exploited in the design of new inhibitors. The discovery that metformin targets this enzyme also strongly supports the proposition that new FBPase inhibitors may have potential as antidiabetic agents. Understanding these communication pathways between two separated sites (the AMP binding and the active site) in the regulation of the enzyme’s overall activity may also be important in determining the molecular changes which underpin FBPase deficiency. It is possible that some point mutations may affect this allosteric regulation, rather than the activity, of the enzyme. In cancers where lack of FBPase activity contributes to tumour progression, there may be a case for introducing the protein using strategies similar to those employed in enzyme replacement therapy for inherited metabolic diseases. Ideally, the protein used would be engineered for increased stability, activity and half-life. The M248D variant may be useful in this context. Finally, many enzymes exhibit allostery and insights gained in relatively well-characterised systems such as FBPase may well have applications in more poorly characterised or newly discovered enzymes.


Results indicated that their qualities such as appearance, flavor, and taste were not significantly different. The low-fat ice cream had higher smoothness scores and lower mouth-coating scores. Hence, pectin extracted from citrus canning wastewater can be potentially used as fat replacer in ice cream, which benefits both the environment and the food industry.Pectin had been recovered from canning wastewater produced by chemical treatment of segment membrane during preparation of canned citrus in our previous research. The purpose of this study was to characterize the extracted pectin from canning wastewater, and to evaluate its application as a fat alternative to replace fat in ice cream. The monosaccharide composition and rheological properties of the pectin were determined. The influences of fat reduction and pectin addition on the physicochemical, rheological and sensory properties of low-fat ice cream were determined. The rheological results showed that pectin solutions were typical pseudoplastic fluids. The addition of pectin in ice cream can cause an increase in viscosity, overrun, and hardness, and a decrease in meltdown of the ice cream. When 0.72% pectin ( China is one of the largest citrus-producing countries in the world and provides nearly 70% canned citrus segments for the international market . Among tin China . The wasd-galactose or 1,5-α-linked l-arabinose. Some of the C-6 carboxyl units of the galacturonic acid backbone are esterified with methoxyl groups or exist as uronic acid salt [Pectin is a heteropolysaccharide block co-polymer comprising 1,4-α-linked galacturonic acid and 1,2-linked rhamnose with side chains of either 1,4-linked β-cid salt . Pectin cid salt . Findingcid salt ,8. Ice cream has been identified as a three-phase made up of a network of fat globules and ice crystals dispersed in a high-viscosity aqueous phase . As one In our previous study, a pilot plant-scale system has been established to recover pectin from processing wastewater . TherefoConsidering the high yield of pectin and the maximum utilization of both acid and alkaline wastewater from citrus canning, the pH of their mixtures was adjusted to the ranges of 3 to 4 before precipitation. The monosaccharide composition of pectin was determined. The pectin has a GalA content of 42.5 mol %, which was lower than the normal value of commercial pectin (> 65%). This most likely is due to the low extraction temperature . RhamnosThe rheological curves of pectin at different concentrations are shown in η is the apparent viscosity (Pa·s), K (Pa·sn) is the consistency index, γ is the shear rate (s−1) and n (dimensionless) is the flow behavior index. The fit parameters obtained are summarized in n), the more obvious the polymer pseudoplasticity. As shown in n values decreased when the pectin concentrations increased, indicating the increased intertwining and breaking of polymer interactions. In Equation (1), where R2 ≥ 0.97). The results showed that the incorporation of pectin significantly changed the rheological properties of ice cream mixes, strengthened the viscosity development, and enhanced the shear thinning behavior as depicted by the increase of K values and the decrease of n value, respectively. Adding hydrocolloids capable of absorbing water and forming a gel-like network in addition to the other components of the mixes would obviously modify its rheology [n-values in this study (0.25–0.79) was wider than a previous study (0.36–0.50) in which various fat replacers were studied in low-fat ice cream [The flow characteristics of ice cream slurry have great influence on the hardness, meltdown and overrun of ice cream products. Slurry with high viscosity can lead to difficult whipping, while too low viscosity of ice cream slurry produces air bubbles with poor stability that significantly affect quality and structure of finished ice cream products . The rherheology . In the rheology  ice crearheology . The rance cream . The effects of pectin addition and fat reduction on the overrun (a), meltdown (b), and hardness (c) of the ice cream are shown in The statistical analysis results showed that the addition of pectin caused an increase in overrun of the ice cream, with a positive contribution to air incorporation. Incorporating air into ice cream mixes is an important step in the manufacture of ice cream, as it provides a soft texture to the products . Pectin A fast-melting product is unwanted while a too slow rate of melting can also be a defect of ice cream. As depicted in Hardness is an important property of frozen products. On the one hand, ice cream must be hard enough so that it can be stored for a certain time and able to resist pressure. On the other hand, its hardness cannot be too high to affect the taste . The resγ = 0.001–10%) at a fixed angular frequency (1 Hz) was conducted  high-performance liquid chromatography (HPLC) method according to our previous study . BrieflyWaters e2695  with a Zorbax Eclipse XDB-C18 column  was used to perform HPLC analysis at 25 °C. The mobile phases were: solvent A, 15% acetonitrile with potassium phosphate buffer , solvent B, 40% acetonitrile with the same buffer. The elution rate was 1 mL/min, relying on a gradient of B from 0% to 15% in the initial 10 min, then from 15% to 25% in the next 20 min. Detection was with a 2489 UV/Vis Detector  at 250 nm. 2O were all purchased from Sigma-Aldrich . All other chemicals used were of analytical grade.Monosaccharide standards, PMP and Dw/v) were prepared with deionized water and placed at room temperature for 24 h before use.Rheological measurements were carried out using a HAAKE RheoStress 6000 rheometer  with a P60 TiL parallel plate. The temperature was maintained at 25 ± 0.1 °C. To investigate the effects of concentration on rheological behavior of pectin, solutions of different concentrations including 0.5%, 1.0%, 1.5% (w/w). Egg yolks were weighed and whipped up. Milk powder, pectin and sucrose were weighed and dissolved at 60 °C for 30 min. The whipping components and solutions were then mixed, and water was made up according to the formulations. After that, the mixtures were pasteurized at 80 ± 1 °C for 30 s using a water bath plate. Then all mixtures were homogenized individually at 65 °C by a laboratory colloid mill , and then cooled to 20 °C. Sterilized milk was added to the cooled samples to ensure the flavors. All mixtures were aged at 4 °C for 24 h to implement complete hydration of all ingredients. Subsequently, the mixtures were frozen in a batch ice cream freezer  for 30 min. The final ice cream samples were filled into 100 mL plastic cups, hardened at −30 °C for 24 h and stored at −18 °C before further physical and chemical analysis. Skim milk powder , cream (35% fat) , pasteurized milk , sucrose and eggs were purchased from local suppliers. The ice cream formulations are listed in Overrun is usually defined as the volume of ice cream obtained in excess of the volume of the mix. It is usually expressed as “percent overrun”. It is one of the important parameters for determining the quality of ice cream. The overrun (%) of ice cream was determined by comparing the weight of the mixture before and after freezing, and calculated according to Equation (2) :(2)OverrIce cream meltdown is one of the important manifestations of ice cream structure when the ice cream is at ambient conditions. Two factors related to the meltdown are melting of the ice and the collapse of the fat-stabilized foam structure. The meltdown is usually expressed as “percentage” and can be determined following the procedure below. The ice cream initially stored at −18 °C was cut by a cooled knife into a standard block (2 cm × 2 cm × 2 cm) to measure the meltdown. The standard block was then placed on a mesh in an enclosed chamber  that was held at 25 ± 0.1 °C and allowed to melt. A funnel is placed beneath the mesh to collect the melted ice cream, which drains into a beaker on a balance. The mass of melted ice cream is recorded after 40 min, and the melting rate is calculated according to Equation (3).−1.Viscosity of the ice cream mixtures before freezing was measured by HAAKE RheoStress 6000 rheometer  with a P60 TiL parallel plate. The temperature was maintained at 25 ± 0.1 °C. The shear rates were changed from 0.1 to 100 sThe hardness of the ice cream was measured with a TA.XT2i texture analyzer . The test conditions were: temperature (4 °C), cylinder stainless probe (P/5), diameter 5 mm, pre-test speed 8 mm/s, test speed 2 mm/s, post-test speed 8 mm/s, depth of penetration 15 mm, and each sample was measured in triplicate.The dynamic-viscoelastic properties of ice cream by using small amplitude oscillatory shear tests were performed on the hardened ice cream. The linear viscoelastic ranges were determined by amplitude sweep from 0.001% to 100% at a constant angular frequency of 1 Hz. The angular frequency sweep was conducted from 0.1 to 10 Hz at 0.01% deformation  to monitor the changing of storage modulus (G′) and loss modulus (G″) of the samples.A panel of 50 judges from our department at the Sensory Laboratory was invited to carry out the sensory evaluations on ice cream samples. They were selected based on regular consumption of ice cream and time availability. The training session and evaluation process were conducted in individual booths at the Sensory Laboratory under controlled lighting and temperature (25 °C). All samples were coded with random three-digit numbers and the serving orders were also randomized. A structured 9-point hedonic scale was employed for the evaluation of appearance, taste, flavor, smoothness, and mouth-coating of ice cream samples. The samples were rated according to the following criteria: nine for liked extremely, eight liked very much, seven liked moderately, six liked slightly, five neither liked nor disliked, four disliked slightly, three disliked moderately, two disliked very much and one disliked extremely.All experiments were performed in triplicate and the statistical results were presented the average of three independent experiments. Statistical analyses were carried out by using the Statistical Analysis Systems  software package. The results were evaluated by analysis of variance (ANOVA) and Duncan’s multiple range test at significance level of 0.05.The present study showed that canned citrus wastewater can be an alternative source for production of pectin with the potential application as fat replacer for high-fat ice cream. First, the study confirmed that the pectin solutions are typical pseudoplastic fluids. Pectin recovered from canned citrus wastewater can be successfully incorporated into the ice cream to replace part of the fat. In addition, compared to the control ice cream, substitution of fat by pectin increased viscosity, overrun and hardness and reduced meltdown of ice cream. Sensory results indicated that pectin can replace 45% of the total fat of ice cream without changing its appearance, flavor, and taste scores. In addition, the low-fat ice cream had significantly higher smoothness score.


To determine outcome of freehand pedicle screw fixation for dorsal, lumbar and sacral fractures at a tertiary care centre in the developing world.st December 2017. A total of 751 pedicle screws were placed. Incidence and extent of cortical breach by misplaced pedicle screw was determined by review of intra-operative and post-operative radiographs and/or computed tomography.A retrospective review was performed of 150 consecutive patients who underwent pedicle screw fixation from January 1, 2012 to 31Among the total 751 free hand placed pedicle screws, four screws (0.53%) were repositioned due to a misdirected trajectory towards the disc space. six screws (0.79%) were identified to have cause moderate breach while four screws (0.53%) cause severe breach. There was no occurrence of iatrogenic nerve root damage or violation of the spinal canal.Free hand pedicle screw placement based on external landmarks showed remarkable safety and accuracy in our center. The authors conclude that assiduous adherence to technique and preoperative planning is vital to success. Pedicle screw fixation with rod constructs has attained global acceptance for stable spine fixation.3The modern surgeon has excellent aides to enhance accuracy and safety of placement of pedicle screws in the spine which include intraoperative C-arm fluoroscopy, intraoperative computer tomography (CT), and computer-assisted navigation.Freehand pedicle screw placement in the thoracic spine is considered both safe and effective and routinely performed by many spine surgeons.st January 2012to 31st December 2017 with a follow up of six months to one year. A retrospective review was performed of 150 consecutive patients who underwent pedicle screw fixation. A total of 751 pedicle screws were placed. This included patients of either sex, aged between 15 to 70 years. After taking history and doing clinical examination, a preoperative radiological assessment was done by using X rays anterio-Posterior and lateral views, MRI and CT scan with 3-D reconstruction of the lumbosacral spine. Patients above the age of 70 years, non-traumatic fractures, with associated disc herniation, previously operated and those who underwent fixation involving fluoroscopic guidance were excluded.The study was conducted at the department of Neurosurgery at PNS Shifa Hospital in Karachi, Pakistan after obtaining approval from the Institutional Review Board. The duration of study was five years from 1All the screws were inserted with free-hand technique using anatomic landmarks as a guide for an entry site. We used the following entry points: thoracic spine; 3 mm caudal to the junction of the transverse process and the lateral margin of the superior articulating process, and the sagittal trajectory was always orthogonal to the dorsal curvature of the spine at that level as described by Fennel et al, in lumbar spine; the junction of the pars interarticularis, the midpoint of the transverse process and the inferior point of the superior articular facet, in sacral; the infero-lateral margin of the basis of the superior articular process of the sacrumWe collected data on the number of patients, the number of inserted pedicle screws, and parameters of the surgical technique . Incidence and extent of cortical breach by misplaced pedicle screw was determined by review of intra-operative and post-operative radiographs and/or computed tomography.Data was analyzed by using Microsoft Excel. Percentages and frequencies were calculated.There were 107 (71.3%) males and 43 (28.7%) females with a mean age of 34.84 ± 14.6 years. The lumbar spine was the commonest site of placement with 561(74.6%) screws, followed by thoracic in 154(21.4%) and sacral in 36(4%). Among the total 751 free hand placed pedicle screws,12 screws (1.59%) were identified to have cause minor breach out of which nine had lateral breaches and 3 inferior breaches . Minor bThe placement of freehand pedicle screws especially in the dorsal spinal segment can be a challenge due to the varied and complex anatomy of the thoracic vertebrae, with a potential for screw malposition and neurovascular injury.The mean accuracy for placement of the screws in our patient group was 96.6%. Literature search reveals that over 8,000 screws have been placed in different studies with the mean accuracy for placement of the thoracic screws at 93.3% where the pedicle screw accuracy was defined as “having the entire screw contained within the cortices of each respective pedicle”.16Among the total 751 free hand placed pedicle screws, 12 screws (1.59%) were identified to have cause minor breach out of which 9 had lateral breaches and three inferior breaches. Minor breaches didn’t require repositioning and were only identified by postoperative imaging. In a study with a total of 720 screws inserted, 623 screws (86.5%) were perfect and 97 screws (13.5%) were misplaced.Most studies agree on the defined “safe zone” ranging from 2 to 4 mm for pedicle screw breach of the vertebral bodies. This safe zone allows the medial or lateral wall breach by the screw without clinical consequences for the patient.23Increasing number of centres are now shifting towards robotic surgery and several studies comparing freehand conventional techniques for pedicle screw fixation robot-assisted (RA) have been published, but with equivocal results. A meta-analysis of five studies with 257 patients and 1105 screws revealed that there was no difference in the accuracy between robot-assisted and conventional freehand pedicle screw placement at the 0 mm grading criteria , and at 2 mm grading criteria .There was no complication encountered in our series which is consistent with the majority of studies in literature which reported no neurological or vascular complications following freehand placement of thoracic screws. The study by Kim, YW et al. gives the strongest evidence of the safety of this technique with their series of 3,204 screws with the longest follow-up (10 years) without any neurological or vascular complication reported.13Lastly, while freehand thoracic screw placement techniques are universally employed, there is dearth of published studies outlining the technical nuances which is further complicated by various starting points and/or trajectories for each level of the thoracic spine, making the adoption of one technique awkward to the spinal surgeon or trainee.Free hand pedicle screw placement based on external landmarks showed remarkable safety and accuracy in our centre. The authors conclude that experience and assiduous adherence to technique and preoperative planning is vital to success.MJ conceived and designed the study.AA did data collection and manuscript writing.AK did statistical analysis & editing of manuscript.SSB did review and final approval of manuscript.


The inhibitory potentials of efavirenz on the enzyme activities of four major UDP-glucuronosyltransferases (UGTs), 1A1, 1A4, 1A6, and 1A9, in human liver microsomes were investigated using liquid chromatography-tandem mass spectrometry. Efavirenz potently inhibited UGT1A4-mediated trifluoperazine N-glucuronidation and UGT1A9-mediated propofol glucuronidation, with Ki values of 2.0 and 9.4 μM, respectively. [I]/Ki ratios of efavirenz for trifluoperazine N-glucuronidation and propofol glucuronidation were 6.5 and 1.37, respectively. Efavirenz also moderately inhibited UGT1A1-mediated 17β-estradiol 3-glucuronidation, with a Ki value of 40.3 μM, but did not inhibit UGT1A6-mediated 1-naphthol glucuronidation. Those in vitro results suggest that efavirenz should be examined for potential pharmacokinetic drug interactions in vivo due to strong inhibition of UGT1A4 and UGT1A9.Efavirenz is a non-nucleoside reverse transcriptase inhibitor used for the treatment of human immunodeficiency virus type 1 infections. Drug interactions of efavirenz have been reported due to  Metabols (UGTs)  /Ki) is 6.5, the inhibition of efavirenz on UGT1A4-mediated trifluoperazine N-glucuronidation is likely, but remains to be demonstrated in vivo. According to this in vitro data, efavirenz should be used carefully with the drugs metabolized by UGT1A4, such as antifungal drugs  /Ki ratio of efavirenz for UGT1A9-mediated propofol glucuronidation was 1.37, inhibition of glucuronidation of UGT1A9 substrates by efavirenz may be possible, suggesting that efavirenz may be used carefully with drugs metabolized by UGT1A9, such as S-etodolac [The –0.4 μM) ,29 and si, 2 μM)  but was etodolac , entacapetodolac , gaboxadetodolac , retigabetodolac , and scoetodolac , to avoiet al. [Ki value of 17 μM, could reduce azidothymidine glucuronidation by approximately 43% at steady-state Cmax vlaues. Ki values of efavirenz for inhibition of UGT1A4-mediated trifluoperazine N-glucuronidation and UGT1A9-mediated propofol glucuronidation were 2.0 and 9.4 μM, respectively, which were much less than its Ki for UGT2B7-mediated azidothymidine glucuronidation (17.4 μM in our unpublished data). Those in vitro results indicate that efavirenz can potentially inhibit the glucuronidation of drugs catalyzed by UGT1A4 and/or UGT1A9 and therefore should be examined for potential pharmacokinetic drug interactions in vivo due to inhibition of UGT1A4 and UGT1A9.Bélanger et al.  estimateTrichoderma viride), hecogenin, and uridine-5-diphosphoglucuronic acid trisodium salt (UDPGA) were obtained from Sigma-Aldrich . Pooled human liver microsomes (H161) were obtained from BD Gentest Co. . Acetonitrile and methanol (HPLC grade) were obtained from Burdick & Jackson Inc. , and the other chemicals were of the highest quality available.Efavirenz and propofol glucuronide were obtained from Toronto Research Chemicals . 17β-Estradiol, 17β-estradiol 3-glucuronide, 1-naphthol, naphthol glucuronide, propofol, trifluoperazine, alamethicin  of efavirenz were determined with UGT assays in the presence and absence of efavirenz  using pooled human liver microsomes and human cDNA-expressed UGT1A1, UGT1A4, UGT1A6 and UGT1A9 isozymes. The incubation mixtures were prepared in a total volume of 100 μL as follows: pooled human liver microsomes or UGT isozymes , 2 mM UDPGA, 25 μg/mL alamethicin, 10 mM MgCl2, 50 mM tris buffer (pH 7.4), UGT-isoform specific probe substrate , and various concentrations of efavirenz (0–200 μM). Reactions were initiated by the addition of UDPGA, and incubations were carried out at 37 °C in a shaking water bath for 30 min. Reactions were terminated by addition of 100 μL of ice-cold methanol containing internal standard . The incubation mixtures were centrifuged at 13,000 × g for 5 min, and then 40 μL of the supernatant was diluted with 60 μL of water. The aliquot (5 μL) was injected onto an LC/MS/MS instrument. All incubations were performed in triplicate and the mean values were used. The glucuronides produced from UGT isoform-specific substrates were respectively determined by LC/MS/MS [N-glucuronide, +5.0 kV; electrospray voltage for 17β-estradiol 3-glucuronide, 1-naphthol glucuronide, and propofol glucuronide, −4.0 kV; vaporizer temperature, 420 °C; capillary temperature 360 °C; sheath gas pressure, 35 psi; and auxiliary gas pressure, 10 psi. Quantification was performed by selected reaction monitoring (SRM) and SRM transitions for the metabolites are summarized in ® software (Thermo Fisher Scientific).The inhibitory potencies , 2 mM UDPGA, 25 μg/mL alamethicin, 10 mM MgCl2, and various concentrations of efavirenz in 50 mM Tris buffer (pH 7.4) in a total incubation volume of 100 μL. Reactions were initiated by addition of UDPGA at 37 °C and stopped after 30 min by placement of the incubation tubes on ice and addition of 100 μL ice-cold methanol containing an internal standard described above. The incubation mixtures were centrifuged at 13,000 × g for 5 min, followed by dilution of 40 μL of the supernatant with 60 μL of water. The aliquot (5 μL) was analyzed by LC/MS/MS.50 values  were calculated using WinNonlin software, a non-linear regression analysis program . The apparent kinetic parameters for inhibitory potential  were estimated from the fitted curves using Enzyme Kinetics Ver. 1.3 program .The ICin vitro human liver microsomes. UGT1A4-mediated trifluoperazine N-glucuronidation and UGT1A9-mediated propofol glucuronidation activities were potently inhibited by efavirenz during incubation with UDPGA in microsomes. Efavirenz also inhibited UGT1A1-mediated 17β-estradiol 3-glucuronidation in a dose-dependent manner but did not inhibit UGT1A6-mediated 1-naphthol glucuronidation. Those results suggest that efavirenz has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by UGT1A4 and UGT1A9. However, the clinical relevance of the inhibitory interaction of efavirenz with UGT1A4- and UGT1A9-substrate drugs has not been investigated. Clinical trials to evaluate the inhibitory effects of efavirenz on UGT1A4 and UGT1A9 remain to be conducted.The effect of efavirenz on four UGTs, 1A1, 1A4, 1A6, and 1A9, was evaluated across a wide range of substrate and efavirenz concentrations using


We investigated the anti-Alzheimer disease (anti-AD) potential of these coumarins by assessing their ability to inhibit acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1). Among these coumarins, 2 exhibited poor inhibitory activity against AChE and BChE, and modest activity against BACE1. Structure–activity relationship analysis showed that 2 has an aldehyde group at the C-6 position, and exhibited strong anti-AD activity, whereas the presence or absence of an aldehyde group at the C-8 position reduced the anti-AD activity of 3 and 1, respectively. In addition, 2 exhibited concentration-dependent inhibition of peroxynitrite-mediated protein tyrosine nitration. A kinetic study revealed that 2 and 3 non-competitively inhibited BACE1. To confirm enzyme inhibition, we predicted the 3D structures of AChE and BACE1, and used AutoDock 4.2 to simulate binding of coumarins to these enzymes. The blind docking studies demonstrated that these molecules could interact with both the catalytic active sites and peripheral anionic sites of AChE and BACE1. Together, our results indicate that 2 has an interesting inhibitory activity in vitro, and can be used in further studies to develop therapeutic modalities for the treatment of AD.Coumarins, which have low toxicity, are present in some natural foods, and are used in various herbal remedies, have attracted interest in recent years because of their potential medicinal properties. In this study, we report the isolation of two natural coumarins, namely umbelliferone ( In addition, hydrophobic interactions between umbelliferone and Lys75, Trp76, Val69, Phe108, and Ile118 were observed with a binding energy of −5.4 kcal/mol. In contrast to 1, the top binding sites of 2 and 3 were distant from the catalytic site, but similar to those of PMF. As shown in 2 with a binding energy of −7.2 kcal/mol. Moreover, hydrophobic interactions between 2 and Ser10, Gly11, Tyr14, Val170, Thr232, Arg307, Pro308, Ala335, and Glu339 of BACE1 also seemed important for binding to the allosteric site, based on inspection of our AutoDock 4.2 model. The molecular docking model of 3 is illustrated in 3–BACE1 complex, Ser10, Gly11, Gly13, Tyr14, Val170, Thr232, Arg307, Ala335, Val336, and Glu339 participated in hydrophobic interactions with 3 without any hydrogen bonding interactions.Molecular docking models of coumarins, 2-amino-3-{ resulted in significant dose-dependent inhibition of ONOO−-mediated tyrosine nitration.To determine the inhibitory effect of Medicinal plants have long provided a reliable source of new drugs to combat diseases. There have also been new trends in the preparation and marketing of drugs based on medicinal plants. Their scientific and commercial significance appears to be gathering momentum in health-relevant areas. Plant-derived products are carefully standardized, and medicinal plants offer several options to modify the progress and symptoms of AD ,26.AD presents as the progressive, inexorable loss of cognitive function associated with the presence of senile plaques in the hippocampal area of the brain. The major hurdle in understanding AD is lack of knowledge regarding the etiology and pathogenesis of selective neuron death. The two most common hypotheses used to explain the pathology of AD are the “cholinergic hypothesis” and “amyloid hypothesis.” The cholinergic hypothesis suggests that AD is caused by a deficiency at the brain level of the cerebral neurotransmitter acetylcholine (ACh), which is hydrolyzed by AChE ,28. SimiIn recent years, considerable data has accumulated that indicates that brains with AD are under increased oxidative stress. This stress may have a role in the pathogenesis of neurodegeneration and death due to AD. Oxidative stress refers to undue oxidation of biomolecules, and often leads to cellular damage. Histopathological and experimental evidence support the significant impact of oxidation on the pathogenesis of AD ,36,37.Many researchers over the past few decades have focused their efforts on designing ChEs and BACE1 inhibitors. However, efforts to discover naturally occurring ChEs and BACE1 inhibitors have been relatively limited. Several plant-derived ChEs and BACE1 inhibitors, including coumarins, anthraquinones, triterpenoids, lignin, flavonoids, and alkaloids ,39,40,412), a benzopyrone-type coumarin, was isolated for the first time from A. decursiva, and showed activity against electric eel AChE, horse serum BChE, and human recombinant BACE1. We assumed that the inhibitory activity of 2 was mainly due to the formyl moiety at the C-6 position, therefore, we further synthesized 8-formyl umbelliferone (3) to determine structure–activity relationships (SARs). To establish SARs between the three coumarins and the target enzymes, the inhibitory effects of the three coumarins against electric eel AChE, horse serum BChE, and human recombinant BACE1 were investigated. 2 and 3 inhibited AChE and BChE with an IC50 range of 16.70–19.13 µM for AChE and 27.90–87.67 µM for BChE, whereas umbelliferone (1) showed weak inhibitory activity. In addition, 2 strongly inhibited BACE1 with an IC50 of 1.31 µM. Among the molecules investigated, both 2 and 3, which have a free aldehyde group at the 6 or 8 position, significantly inhibited ChEs and BACE1. A comparison of the three coumarins indicated that the presence of an aldehyde group markedly increased inhibitory activity  and PAS (Tyr334) of AChE, whereas 3 bound only to CAS (His440). In the BACE1 docking simulation, we observed multiple hydrogen bonds in the docked BACE1–2 complex. However, 3 did not form any hydrogen bonds with BACE1, even though it docked in a similar region to 2. These docking data showed that hydrogen bonds between the coumarins and major active residues of the target enzyme play a crucial role in enzyme inhibition. Moreover, we also confirmed the inhibitory activity of 2 against formation of ONOO− mediated tyrosine nitration by Western blotting.Computational molecular docking analysis can provide insight into the mechanism underlying active site binding interactions. 2 has significant potential to inhibit, and may prevent AD by targeting Aβ formation through inhibition of BACE1 and AChE, as well as ONOO−-mediated tyrosine nitration.Taken together, the in vitro results and in silico molecular docking data indicate that 1H- and 13C-NMR spectra were measured using a JEOL JUM ECP-400 spectrometer  at 400 MHz for 1H-NMR and 100 MHz for 13C-NMR, with the compounds dissolved in deuterated dimethyl sulfoxide (DMSO-d6) and chloroform (CDCl3). Column chromatography was carried out using 70–230 mesh silica gel . Thin-layer chromatography (TLC) was performed on pre-coated Merck Kiesel gel 60 F254 plates (0.25 mm), and 25% H2SO4 was used as a spray reagent. All solvents for column chromatography were of reagent grade and were acquired from commercial sources.− was purchased from Molecular Probes Cayman . All other chemicals and solvents used were purchased from E. Merck, Fluka, or Sigma- Aldrich, unless otherwise stated.Electric eel acetylcholinesterase , horse serum butyrylcholinesterase , acetylthiocholine iodide (ACh), butyrylthioline chloride (BCh), 5,5′-dithobis [2-nitrobenzoic acid] (DTNB), ethylenediaminetetraacetic acid (EDTA), berberine, and quercetin were purchased from Sigma-Aldrich Co. . The BACE1 FREF assay kit (β-secretase) was purchased from Pan Vera Co. . ONOOA. decursiva plants was refluxed with methanol (MeOH) for 3 h (3 × 10 L). The total filtrate was then concentrated until dry in vacuo at 40 °C to render the MeOH extract. This extract was suspended in distilled water and then successively partitioned with dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and n-butanol (n-BuOH) to yield CH2Cl2, EtOAc, n-BuOH, and H2O fractions, respectively, according to Zhao et al. [2Cl2–MeOH  to obtain 20 subfractions (F-1 to F-20). Repeated chromatography of subfraction-6 over a silica gel column using CH2Cl2–MeOH  yielded 6-formyl umbelliferone (2) (30 mg) and umbelliferone (1) (3.9 g), respectively. The structure of the compounds was confirmed by 1H- and 13C-NMR spectroscopy and comparison with published data [Powder of whole o et al. . Initialhed data ,23. Stru6-Formyl umbelliferone (2). Colorless needles; 1H-NMR : δH 11.7 , 10.24 , 8.03 , 8.04 , 6.84 , 6.32 ; 13C-NMR : δC 189.2 (-CHO), 163.4 (C-7), 159.5 (C-2), 158.7 (C-9), 144.4 (C-4), 130.0 (C-5), 120.4 (C-6), 113.1 (C-3), 111.96 (C-10), 103.4 (C-8).3) was prepared from umbelliferone  and urotropine  in an ice-bath, as described by Qiao et al. [n-hexane/EtOAc = 10:1, v/v) to yield 3 as a light yellow powder .8-Formyl umbelliferone . Light yellow powder, 1H-NMR : δ 12.22 , 10.61 , 7.66 , 7.60 , 6.89 , 6.33 , 13C-NMR : 192.94 (-CHO), 165.51 (C-7), 159.12 (C-2), 156.74 (C-9), 143.36 (C-4), 135.98 (C-5), 114.70 (C-10), 113.42 (C-8), 110.87 (C-3), 108.68 (C-6).1–3 and 2–50 µM for quercetin.A BACE1 fluorescence resonance energy transfer (FRET) assay kit  was purchased from PanVera Co. . The assay was carried out according to the provided manual with slight modifications, as described in a previous study . Quercet1–3 and the positive control (berberine) were dissolved in 10% DMSO. The tested concentration range was 4–250 µM for 1–3 and 0.32–20 µM for berberine. Reactions were initiated upon addition of 10 µL of DTNB and 10 µL of either ACh or BCh. Enzymatic hydrolysis, mediated by AChE or BChE, was monitored according to the formation of yellow 5-thio-2-nitrobenzoate anions at 412 nm for 15 min, which were generated by the reaction of DTNB with thiocholine released from ACh or BCh. All reactions were performed in 96-well plates in triplicate, and recorded using a microplate spectrophotometer .The inhibitory activity of the isolated coumarins toward ChE was measured using the spectrophotometric method developed by Ellman et al. . ACh and2 in BACE1 inhibition kinetic experiments were 0, 0.4, 2.0, and 10 µM, while those for 3 were 0, 25, 50, and 100 µM. Inhibition constants (Ki) were determined by interpreting the Dixon plot, where the value on the x-axis represents Ki [Complementary kinetic methods were employed: Lineweaver–Burk and Dixon plots ,51. The sents Ki .http://www.rcsb.org/), with the respective accession codes 1acj and 2wjo, respectively. The co-crystallized ligands, THA and QUD, were used to generate the grid box for a catalytic inhibition mode. The reported allosteric inhibitors, donepezil and PMF, were also used to compare the interaction residues [1–3 were drawn with Chemdraw Ultra 12.0 , and their pKa values were computed at crystallographic pH (pH = 7.5) using the MarvinSketch . Docking simulation of 1–3 with AChE and BACE1 were performed individually using AutoDock Tools (ADT), and all torsions were allowed to rotate. The grid box size was 40 × 40 × 40 with a default spacing of 0.375 Å, and the x, y, z, center was 6.312, 71.239, 69.019 for AChE, and 18.167, 36.716, 40.55 for BACE1. The docking parameters were used as defaults of the ADT and Lamarckian genetic algorithm method was employed. The results were analyzed using PyMOL 1.7.4 and Ligplot+.Molecular docking was carried out with AutoDock 4.2 program using the default parameters, with slight modification . The tarresidues ,54, and −-mediated protein tyrosine nitration using the method of Ali et al. [2 was 12.5–100 µM.We evaluated ONOOi et al. , with slt-test where appropriate . Values of p < 0.05 were considered statistically significant.All results are expressed as means ± SEMs of triplicate samples. Results were analyzed using one-way ANOVA and Student’s 2, which has an aldehyde group at 6 position, inhibited BACE1 non-competitively with lower iK and IC50 values compared to 1 and 3. In addition, the 3D molecular docking study showed that 2 docked into the allosteric site of BACE1. These modeling results were consistent with our in vitro findings, which revealed that inhibitory activities against BACE1 are closely related to the presence of the formyl moiety at position 6 in 2. Moreover, 2 exhibited potent inhibitory activity against ONOO−-mediated tyrosine nitration, which is one of the important therapeutic targets in AD. From the results of the present study, we can conclude that introduction of the formyl moiety to 1 increases the inhibitory activity against ChEs and BACE1, and that its location at position 6 (as in 2) is more effective that at position 8 (as in 3). To the best of our knowledge, this is the first report of the potential anti-AD activity of 2 and 3. Therefore, 2 could play an important role in the development of new therapeutic drugs to treat AD. Further studies are needed to determine if 2 has the same effects in vivo and to clarify the underlying mechanisms.The present study revealed that coumarins may have significant anti-AD activity through inhibition of ChEs and BACE1 in the Aβ pathway. In particular,


Collapse of the West Antarctic Ice Sheet can in principle be stopped by artificial snowfall but challenges are large and manifold. −1 sea level equivalent). Mass deposition at a lower rate increases the intervention time and the required total amount of snow. We find that the precise conditions of such an operation are crucial, and potential benefits need to be weighed against environmental hazards, future risks, and enormous technical challenges.There is evidence that a self-sustaining ice discharge from the West Antarctic Ice Sheet (WAIS) has started, potentially leading to its disintegration. The associated sea level rise of more than 3m would pose a serious challenge to highly populated areas including metropolises such as Calcutta, Shanghai, New York City, and Tokyo. Here, we show that the WAIS may be stabilized through mass deposition in coastal regions around Pine Island and Thwaites glaciers. In our numerical simulations, a minimum of 7400 Gt of additional snowfall stabilizes the flow if applied over a short period of 10 years onto the region (−2 mm year From the mid-1990s to the year 2010, the ice loss from both regions substantially increased from 6 Gt yearMost climate models indicate an increase in snowfall over Antarctica under future global warming . The model applies a superposition of the shallow ice  of the ice sheet in the region adjacent to the grounding line. The enforced increase in ice surface elevation upstream of the grounding line induces a strong steepening of the surface slope, enhancing the driving stress at the grounding line area . The stabilization threshold curve Rc, which we fit to the data obtained from our simulations, is indirectly proportional to the perturbation duration T with an offset Roff. Calculation of the total minimum amount of mass that has to be deposited (Mc= Rc · T) transforms this threshold toFrom our ensemble of perturbation experiments, we infer the critical mass addition rate ration T : High  = infinity to Mc(R → ∞) = M0. Together, This critical threshold decreases nonlinearly with increasing mass addition rate R , asymptoOur model simulations neglect several mechanisms that could alter the speed and degree of the potential ice sheet collapse, including possible snowfall increase and ice shelf hydrofracturing due to future atmospheric warming, the effect of glacial isostasy, or consequences of marine ice cliff failure , a power that, in theory, could be provided by more than 12,000 high-end wind turbines driven by the regional wind fields, which in principle would have sufficient capacity . In our simulations, this area covers the joint coastal region of PIG and TG with an extent of about 52,000 km17 m·s and a minimum ice thickness at the calving front of 200 m. The physically motivated calving law takes into account the eigenvalues of the horizontal strain-rate tensor, allowing for geographically confined ice shelves and dynamic calving front positions. A physical stress boundary condition was applied at the calving front to close the equations of the shallow shelf approximation . We Initial bed and ice topography was obtained from the Bedmap2 dataset (


Epimedium koreanum are presumed to be due to the flavonoid component icariin, which has been reported to have broad pharmacological potential and has demonstrated anti-diabetic, anti-Alzheimer’s disease, anti-tumor, and hepatoprotective activities. Considering these therapeutic properties of icariin, its deglycosylated icaritin and glycosylated flavonoids  were evaluated for their ability to inhibit protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase. The results show that icaritin and icariside II exhibit potent inhibitory activities, with 50% inhibition concentration (IC50) values of 11.59 ± 1.39 μM and 9.94 ± 0.15 μM against PTP1B and 74.42 ± 0.01 and 106.59 ± 0.44 μM against α-glucosidase, respectively. With the exceptions of icaritin and icariside II, glycosylated flavonoids did not exhibit any inhibitory effects in the two assays. Enzyme kinetics analyses revealed that icaritin and icariside II demonstrated noncompetitive-type inhibition against PTP1B, with inhibition constant (Ki) values of 11.41 and 11.66 μM, respectively. Moreover, molecular docking analysis confirmed that icaritin and icariside II both occupy the same site as allosteric ligand. Thus, the molecular docking simulation results were in close agreement with the experimental data with respect to inhibition activity. In conclusion, deglycosylated metabolites of icariin from E. koreanum might offer therapeutic potential for the treatment of type 2 diabetes mellitus.The therapeutic properties of  O-linkage-hydrolases that release D-glucose from the non-reducing substrate end -1-naphthyl}-l-alanyl)amino]pentyl}oxy)-2-naphthoic acid  and 3--2-ethyl-benzofuran-6-sulfonic acid (4-sulfamoyl-phenyl)-amide  were considered as the standard ligands to which the orientation of the tested compounds were compared. Docking analyses were conducted to determine the extent of agreement of the structure-activity compound relationships with the obtained experimental data, as well as to evaluate binding site-directed inhibition of PTP1B. The AutoDock 4.2 program -1-naphthyl}-l-alanyl)amino]pentyl}oxy)-2-naphthoic acid (compound 23) were obtained from the RCSB Protein Data Bank [The X-ray crystallographic structure of PTP1B complexed with its selective inhibitor 3--2-ethyl-benzofuran-6-sulfonic acid (4-sulfamoyl-phenyl)-amide .All results are expressed as the mean ± SEM of triplicate samples. Statistical significance was analyzed using one-way ANOVA and Student’s E. koreanum and their constituents possess anti-DM properties. Comparative evaluations of these activities of E. koreanum might be useful in the development of therapeutic agents for treating patients with DM. Among the tested compounds, icaritin and icariside II (two deglycosylated metabolites of icariin) significantly inhibited PTP1B. Furthermore, enzyme kinetics analysis and molecular docking simulations supported these results. Thus, approaches combining computational work with experimental validation are useful for developing new natural drug candidates against diabetes. These active candidates have significant potential as drug candidates for treating DM. Further investigations of the bioactivity of these natural products are needed to determine the precise therapeutic and/or preventive potential of E. koreanum and its constituents in vivo.The results of the current study indicate that the aerial parts of


Cyclin-dependent kinases (CDKs) coordinate hundreds of molecular events during the cell cycle. Multiple cyclins are involved, but the global role of cyclin-specific phosphorylation has remained unsolved. We uncovered a cyclin docking motif, LxF, that mediates binding of replication factor Cdc6 to mitotic cyclin. This interaction leads to phospho-adaptor Cks1-mediated inhibition of M-CDK to facilitate Cdc6 accumulation and sequestration in mitosis. The LxF motif and Cks1 also mediate the mutual inhibition between M-CDK and the tyrosine kinase Swe1. Additionally, the LxF motif is critical for targeting M-CDK to phosphorylate several mitotic regulators; for example, Spo12 is targeted via LxF to release the phosphatase Cdc14. The results complete the full set of G1, S, and M-CDK docking mechanisms and outline the unified role of cyclin specificity and CDK activity thresholds. Cooperation of cyclin and Cks1 docking creates a variety of CDK thresholds and switching orders, including combinations of last in, first out (LIFO) and first in, first out (FIFO) ordering.  •Mitotic cyclin Clb2 binds a specific linear motif, LxF, in targets or inhibitors•LxF interaction enhances mitotic CDK substrate phosphorylation•Phospho-adaptor Cks1 and the LxF docking mediate CDK inhibition by Cdc6 and Swe1•Cyclin-specific targeting enables finetuning of CDK function Örd et al. find that a short linear motif in proteins enables specific targeting by the mitotic CDK. Depending on the context, this motif can lead to either enhanced phosphorylation of mitotic targets or inhibition of the CDK complex in cooperation with the phospho-adaptor subunit Cks1. To accomplish one of the most complex biological tasks, the assembly of an extra copy of the cell, cyclin-dependent kinases (CDKs) coordinate hundreds of events during the cell cycle . In thisAlthough it is well established that the cell cycle is coordinated by CDK, it has been difficult to outline a systems-level mechanism for the temporal organization of CDK-driven events. Besides a very coarse-grained categorization of early, mid, and late targets , cyclin In addition, experimental evidence suggests that cyclin-specific substrate recognition also controls the temporal order of CDK-driven switches. Cyclins affect CDK specificity in two ways. First, it was found in yeast and then in mammals  that cycThe mitotic cyclins have several specific functions. In budding yeast, the mitotic cyclins Clb2 and Clb1 initiate anaphase and promote the isotropic growth switch in early mitosis and spindle elongation during anaphase . AdditioPreviously, we found an example of extreme cyclin specificity that sharply discriminates between the closely related B-type cyclins of budding yeast, the Clbs . Cdc6, aPhosphorylation of Cdc6 determines the time window for replication origin licensing . PhosphoIn the present study, we dissected the mechanism of Cdc6-mediated inhibition and present a unique inhibitory mechanism that involves an M-CDK-specific cyclin docking motif, LxF, in Cdc6 and the phospho-adaptor Cks1, that leads to shielding of the degron and sequestration of Cdc6 by M-CDK during mitotic exit. In addition, the M-CDK docking motif was found to play a broader role in CDK function during mitosis. The docking motif is essential for phosphorylation of Spo12 and activation of the fourteen early anaphase release (FEAR) network; it targets M-CDK activity during the isotropic growth switch, directs Clb2 localization to the bud neck, and enables specific regulation of M-CDK by Swe1. Finally, we show that the CDK threshold and the cyclin specificity models are not alternatives; instead, our data lead to a unified model of CDK function according to which cyclin specificity and Cks1 mechanisms provide flexibility for creating many CDK thresholds and complex temporal switching orders.hp), and mutation of the pocket (hydrophobic patch mutant [hpm]) reduced the phosphorylation rate. In contrast, a mutation in the hp of M-cyclin enhanced the phosphorylation of Cdc6. Such a remarkable cyclin-specific phosphorylation profile suggested that the interaction between Cdc6 and M-CDK could be inhibitory, whereas the S-CDK hp interaction promotes phosphorylation. Indeed, M-CDK was inhibited by Cdc6 with low nanomolar Ki values, whereas inhibition was weakened by more than two orders of magnitude in the hpm version  and Clb2 (M-CDK) complexes, Cdc6 was efficiently phosphorylated by both Clb5 (S-CDK) and Clb3 (G2-CDK) complexes A. Phosph version A.To map the critical elements in Cdc6 responsible for inhibition, we first analyzed the disordered N terminus D. Deletihp of Clb2. Judging from the known distance requirements between the Cks1 binding sites, the active site, and the hp  was used instead of Clb2(wild-type [WT]). On the other hand, the mutation 126FQSLP130 reduces inhibition, even in the case of Clb2(hpm), suggesting that LxF binds to the hp ) reduced inhibition by more than 100-fold, whereas mutations in Cks1 and phosphorylation sites caused only a 10-fold reduction, we reasoned that the remaining affinity depends on a docking motif that binds to the d the hp , we idenhibition B. Howevehibition E. Additihibition G. The efo the hp E. Therefhibition H.CLB2-Citrine and CDC6-Citrine (CDC6 was replaced by cdc6(lxf), which contains the mutated docking motif, the Cdk1 activity sensor showed only a 1-min delay in nuclear accumulation  mutation did not lead to a further decrease. These results agree with previous reports suggesting that Cdc6, Sic1, and Cdh1 cooperatively suppress M-CDK in mitotic exit . In the case of wild-type Cdc6, M-CDK phosphorylated mainly one site in Cdc6, and addition of S-CDK did not lead to further phosphorylation , showing a similar profile as Cdc6(T39A S43A T368A S372A lxf), where both degrons are mutated  strain  phenotype in the sensor localization (clb2(hpm) effect on sensor dynamics is not due to the lack of Cdk1 inhibition but, instead, the absence of LxF-mediated M-CDK activity and, thus, a lower level of mitotic phosphorylation. To find the key targets that cause this effect, we analyzed the disordered regions of mitotic Cdk1 targets and introduced mutations in a number of candidate LxF motifs.We found that nuclear entry of the Cdk1 activity sensor was severely delayed in a ) strain A–4C. Suc in Cdc6 E, raisedlization A–4C. Thihp and a conserved LxF motif , a similar drop in phosphorylation as with the LxF1 mutant was observed  A. A kinaobserved A. These in vivo, we set up an assay to measure Swe1-mediated mitotic arrest because of depolymerization of actin in response to treatment with Latrunculin B (Lat B) (in vivo (swe1(T196S) strain was considerably less hindered upon actin depolymerization compared with the wild-type strain (To test the role of the Swe1 candidate LxF motifs  (Lat B) . Cells w (Lat B) C. Both t (Lat B) B. This i or bni1(lxf) cells, GFP-Bud6, although still detectable at the bud tip, was localized to the bud neck in less than 60% of metaphase cells (clb2(hpm)- and bni1(lxf)- expressing cells have slightly elongated buds in the GFP-BUD6 strain  or clb2(hpm) (Bud3 has been shown to recruit Clb2 to the bud neck in a  of Swe1 . To testlb2(hpm) B–S6D.cat/KM) of Cdk1 in an increasing order corresponding to their appearance in the cell cycle: G1-CDK < S-CDK < G2-CDK < M-CDK . This motif was first mapped in the replication licensing factor Cdc6 while exploring its contribution to an inhibitory mechanism initiated via priming of Cdc6 for the phospho-adaptor Cks1. This mechanism leads to shielding of the Cdc6 N-terminal degron by M-CDK, which would also prevent Cdc6 from loading replication origins before G1, as shown previously by hp of cyclins, hp being different but still conserved among M-cyclins. The work presented here has shown that, indeed, M-CDK has minor specificity toward the S-CDK specific RxL motif; however, it has evolved to bind a different M-CDK specific motif , which is used in transcriptional networks to order the sequential activation of genes given the gradual accumulation of a common input signal . A simplmart.loog@ut.ee).Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Mart Loog -RP cells that were grown in 2xYT media.Yeast strains were haploid derivates of the W303 strain and are described in In studies that analyzed the LxF motif in substrate docking we used substrate constructs based on the N terminus of Sic1. The Sic1 sequence from positions 1-33 with mutations T2A and T5S followed by a linker with sequence ELQGGGGG was fused with immunoglobulin-binding domain of streptococcal protein G (GB1 domain) containing a C-terminal 6xHis tag. In constructs with cyclin docking motifs, amino acids in positions 27-33 were replaced with either the sequence PEKLQF from Cdc6 (LxF motif) or with the sequence VNRILFP from Sic1 (RxL motif).Clb5-, Clb3-, Clb2-Cdk1 complexes and full-length Swe1 were purified from yeast cells using TAP method with C-terminally tagged cyclins as described previously , except E. coli BL21RP at 23°C using 0.3 mM IPTG. Sic1-GB1-6xHis-tag substrate constructs were expressed in E. coli BL21RP at 37°C using 1 mM IPTG. His-tagged proteins were purified by standard cobalt affinity chromatography with 200 mM imidazole used for elution. GST-Spo12, GST-Bud3(1222-1636) and GST-Bni1(1215-1953) were expressed in E. coli BL21Rosetta at 18°C using 0.3 mM IPTG and were purified using glutathione-Sepharose beads . Cells were lysed using lysozyme, except in case of Swe1(1-450), the lysate was prepared using Mixer Mill MM 400 (Retch).N-terminal 6xHis-tag was used for purification of Cdc6, Swe1(1-450) and Fir1. 6His-tagged substrates were expressed in 2, 20 mM imidazole, 2% glycerol, 0.2 mg/ml BSA, 500 nM Cks1 and 500 μM ATP .The general composition of the assay mixture contained 50 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM MgClTo analyze the inhibition of Cdk1 activity by Cdc6, we used bovine histone H1 (Sigma-Aldrich) as a reference substrate . In the inhibition assays, we used 7-10 reactions with Cdc6 concentrations ranging from 0.1 nM to 3 μM and two reactions containing no Cdc6. The concentration of the Clb2-Cdk1 complex was 0.2 nM and reactions were started by mixing the enzyme with a solution containing Cdc6 and histone H1. Reactions were carried out at room temperature and were stopped using SDS-PAGE sample buffer at the 12-minute time point.0 range, several-fold below the estimated KM value). The concentrations of kinase complexes were 0.2-2 nM. The kinase assays were performed under conditions below 10% of the initial substrate turnover. Reactions were stopped using SDS-PAGE sample buffer.For the phosphorylation assays, substrate protein concentrations were in the range of 1-2 μM  and quantification of signals was performed using ImageQuant TL (Amersham Biosciences). GraphPad Prism 5.0 was used for data analysis.Yeast cultures were grown at 30°C in synthetic complete media with 2% glucose (SC) to OD 0.2-0.6. Cells were pipetted onto 0.8 mm coverglass and covered with a 2% agarose pad made with SC. Before imaging, the cells were allowed to proliferate on the slide for 60 min. Imaging was performed at 30°C using a Zeiss Observer Z1 microscope with a 63X/1.4NA oil immersion objective and Orca-r2 C10600-10B camera (Hamatsu Photonics). Temperature of the agarose pad was held at 30°C using Tempcontrol 37-2 digital from PeCon. Images were taken every 3 min and imaging sessions were 8 h long. Up to 12 positions were imaged using an automated stage and Axiovision software. Definite Focus was used to keep the cells in focus during time-lapse microscopy. NLS-NES-GFP expressing strains were exposed for 15 ms using a Colibri 470 LED module. Clb2- and Cdc6-yeCitrine strains were exposed for 250 ms using Colibri 505 LED module. Spc42-mCherry was imaged using Colibri 540-580 LED module for 750 ms. All Colibri modules were used at 25% power. Image segmentation, cell tracking and quantification of nuclear fluorescence signals was performed using MATLAB  as described in Spo12-13MYC expressing cells were grown in YPD at 30°C to OD 0.3, then treated for 2.5 hr with 1 μg/ml α-factor and released into fresh medium. Cells were lysed by bead beating in a lysis buffer containing urea. For analysis of Spo12 phosphorylation, proteins were separated using Phos-tag SDS-PAGE with 50 μM Phos-tag and 8% acrylamide. Blotting of Phos-tag SDS-PAGE gels was performed using a dry system iBlot (Invitrogen). Blotting of SDS-PAGE gels was carried out using a Pierce G2 Fast Blotter (Thermo Scientific). c-Myc monoclonal antibody (1:500)  and HRP-conjugated anti-mouse antibody (1:7500) from Labas, Estonia were used to detect MYC-tagged proteins by western blotting. To detect Clb2 protein, we used rabbit polyclonal Clb2 antibody (1:500)  and HRP-conjugated anti-rabbit antibody (1:7500) from Labas, Estonia.Yeast cultures were grown overnight at 30°C in synthetic complete media with 2% glucose (SC) to OD 0.6. Cultures were pipetted into 80% ethanol and cells were fixed overnight in a rotator at 4°C. Fixed cells were treated with RNase A (Sigma) (1 mg/ml RNase A for 40 min at 37°C) and Proteinase K (Thermo Scientific) (200 μg/ml Proteinase K for 1h at 37°C). DNA was stained with SYBR Green I (Sigma-Aldrich) for 1 h.S. cerevisiae proteome for potential LxF docking motifs. The IUPRED disorder cut-off score was 0.3. Based on mutational mapping of the motif in Cdc6 and conservation of the motifs, we searched for 3 motifs: P.[KR]L.F, [NE][KR]L.F and P[NE].L.F (where ‘.’ is any amino acid). The hits from these 3 searches were gathered as potential LxF motifs.SLiMSearch4  was usedhttp://bioinf.cs.ucl.ac.uk/psipred/).Protein secondary structure predictions were performed using PSIPRED v3.3 (The phylogenetical analysis of mitotic cyclins in Figure S7B was done using the Phylogeny.fr platform One-Click pipeline .Structural analysis and alignment of cyclin sequences to human cyclin A2 structure 2CCI;  was carrThe data from time-lapse microscopy experiments is from at least two replicate experiments with different transformants in case of Cdc6 mutant strains. All replicate experiments are included in the data. The statistical details of the experiments can be found in the figure legends, the exact number of cells used in microscopy data is presented in https://doi.org/10.17632/r7hv4zj6zw.1.Unprocessed autoradiographs and western blot images of this study are available at this link: