The culmination of the glaciers in the European Alps during the Last Glacial Maximum (LGM) is one of the most intensively studied paleoglaciological events, but its trigger and forcing remain incompletely understood. Here, we provide evidence that the timing of this glacier maximum coincided within age uncertainties with a 3100 yr-long interval of subsurface warming (26.6 to 23.5 ka BP) as recorded by an archive preserved in caves, cryogenic carbonates. This interval of sustained permafrost degradation during one of the coldest intervals of the last glacial period calls for a fundamental change in the dry Arctic-style precipitation regime. Instead, heavy snowfall during autumn and early winter led to the accumulation of a seasonal snowpack insulating the ground from the winter chill. Combined with thermal modelling, the data provide compelling evidence that the LGM glacier advance in the Alps was fueled by intensive snowfall late in the year, likely sourced from the Mediterranean Sea. What controlled changes of glaciers in the European Alps at the time of their largest extent, about 25,000 years ago, is not well known. Here, the authors use cryogenic carbonates in caves to show that heavy snowfall during autumn and early winter was the main source of glacier growth. Some authors prefer a slightly later onset of the LGM at 26.5 ka BP and an end at 19 ka BP3.The Last Glacial Maximum (LGM) is one of the best documented episodes in the Quaternary history of the Earth marked by a maximum in ice extent on the continent associated with a lowstand in global sea level. The LGM is stratigraphically defined to between 27.5 and 23.3 ka BP5 as well as the chronology of its buildup and decay9 have been thoroughly studied since many decades. Paleoglaciological studies have revealed that the ice divide during the LGM was located south of the modern weather divide, which suggests that prevailingly southerly circulation delivered Mediterranean-sourced moisture to the Alps at this time10. This pattern is in contrast to the modern advection of North Atlantic moisture from the (north)west and indicates that the southward displacement of the polar front caused mid-latitude cyclones to follow a more southerly route across the Mediterranean Sea, consistent with global circulation models showing evidence of a predominantly southerly flow pattern12. This scenario is corroborated by paleobotanical data showing a steep moisture gradient from the southern rim of the Alps  to the northern foreland13. It is also consistent with paleoglacier models of the Alpine ice-stream network14, although one recent model study suggests a predominant moisture advection from the west15. Precisely dated evidence for a southward shift of the North Atlantic storm track during the LGM was reported from a cave system in Switzerland. Oxygen isotope data of stalagmites reveal a marked drop between 26.5 and 23.5 ka BP, coincident with the timing of the maximum ice volume in the Alps. The isotopic signal is consistent with moisture transport from the south across the main Alpine crest16. Although there is now compelling field- and proxy-based evidence that the maximum advance of Alpine glaciers during the LGM was triggered by precipitation associated with predominantly southerly moisture transport to and partly across the Alpine barrier16, it has remained unclear whether this “föhn-type” airflow and precipitation occurred seasonally or year-round.In the European Alps, the cradle of ice age research, the LGM marked the largest glacier extent of the last glacial cycle. Both the geometry of the ice-stream network and its large piedmont glaciersHere, we show that permafrost in the unglaciated Obir region on the southeastern rim of the Alps warmed during the LGM, precisely at the time of the hypothesized southerly circulation. Subsurface warming during full stadial conditions requires a sufficiently thick seasonal snowpack to insulate the ground from the winter cold and therefore calls for a drastic change in the LGM hydroclimate. We argue that enhanced Mediterranean-sourced autumn to early winter snowfall provided the extra precipitation that allowed the Alpine glaciers to expand to their maximum extent for a few thousand years.2-rich groundwater prior to or during the Neogene uplift of the mountain range17. The galleries, chambers, and pits form isolated caves, which can only be accessed by abandoned mining tunnels. The caves show abundant paleophreatic morphologies and lack evidence of later vadose entrenchment by cave streams. Breakdown blocks are wide-spread and none of the galleries extends to the surface except one narrow shaft that was artificially modified by mining activities. This study focusses on two parts of the Obir cave system located between ~1050 and 1100 m a.s.l., the 1.5 km-long Rasslsystem, and the adjacent but unconnected 0.9 km-long Banane system.Underground cavities on this eastern slope of Hochobir (2139 m a.s.l.) in the Northern Karawanks, collectively known as Obir caves . In both caves, which lack perennial ice today, we identified coarse crystalline cryogenic cave carbonates (CCC for short). These carbonate speleothems form by very slow freezing of small water pools created by drip water on perennial cave ice deposits, providing robust 230Th-dated anchor points for thermal conditions very close to 0 °C19. The depths of the caves in which CCC were found range from 42 to 67 m below the surface  and very close to the multi-annual mean of the air temperature outside the caves at this elevationCCC was identified in several chambers and passages in both caves . Detrital contamination is low resulting in fairly accurate and precise ages ranging from 26.6 ± 0.2 ka to 23.5 ± 0.1 ka (Supplementary Data To quantitatively explore the relationship between thawing in the subsurface as recorded by CCC and climate change we applied a 1d heat-flow model (see Methods). In the first scenario, we simulated the initial development of permafrost as a result of a change towards a cold and dry stadial climate. As the starting condition, we assume a ground temperature of 1 °C  and used seasonal temperature values for stadial conditions consistent with regional proxy data . The res2, i.e. 1.1–4.2 ka prior to CCC formation, and lasted only some 200 years. As the initial setup we used the permafrost conditions simulated in scenario 1, i.e., the surface in near thermal equilibrium with the depth of the caves after ~1000 yr of stadial climate. The latter is a realistic figure given that the average duration of stadials in Marine Isotope Stages (MIS) 3 and 2 was at least 1000 yr2. We applied instantaneous atmospheric warming from −4 °C to 2 °C mean annual air temperature (MAAT) to allow the permafrost to thaw, consistent with the rapid warming at the onset of such interstadials as recorded in Greenland ice cores21. Allowing for the warming to last 200 yr, i.e., the duration of GI 32, results in a temperature close to 1 °C at 70 m depth  but allowed for a small amount of winter warming  given that this scenario invokes a significant increase in autumn and early winter precipitation . We set ΔT at 6 °C, i.e., the snow cover attenuates the temperature at the top of the ground in January from −11 °C to −5 °C. This value of ΔT is within the range of modern ΔT observed on the North Slope of Alaska . We explored whether this scenario can explain prolonged permafrost thawing during the long GS 3 as recorded by CCC data. During the preceding GI 3 permafrost likely underwent some thawing and we used the thermal conditions at the end of this 200 yr-long interstadial (scenario 2) as the starting condition. The results show that the depth zone of the caves stays close to 0 °C for an extended period of time, e.g., the 50 m level reaches −0.1 °C after ~150 yr and then approaches thermal equilibrium at −0.5 °C . We used ΔT, the difference between ground temperature and air temperature, to describe the net thermal effect of snow cover on the ground temperature °C Fig. .Fig. 4ScFor the three scenarios presented above, we also considered the thermal effects of ground ice, air advection, and latent heat transfer via seepage water. Although it is likely that they contributed to the rate of temperature change in the subsurface in response to climate change, we rule out that they significantly affected the magnitude of thermal changes as modeled with the conduction-only setup .25, and a delicate setup19. First of all, cave ice of sufficient thickness  must be present. Second, the temperature of the ice and the chamber must be just slightly below 0 °C and the rock above the cave must be close to the melting point allowing the influx of  drip water into the cave26. If the cave temperature is significantly lower than ~−1 °C the water refreezes rather quickly and results in massive deposition of ice either as ice stalactites and stalagmites and/or as thick-bedded floor ice27. If the dripping water encounters floor ice close to 0 °C, however, it will slowly melt the ice close to the impact point forming near-circular pools on the ice. Importantly, these depressions are filled with drip water that contains significant amounts of Ca and bicarbonate ions from the overlying karst rock (as opposed to very dilute ice meltwater). Third, the intra- and inter-annual temperature variations of the chamber and the ice therein must be very small (likely not exceeding a few tenths of a degree), thus allowing for a very slow downward re-freezing of the water in these pools. If the intra-annual temperature amplitude is larger and involves strong winter cooling then the re-freezing would be fast resulting in the precipitation of fine crystalline CCC varieties19. Only very slow freezing of these water pools and pockets in the ice will lead to a progressive increase in ionic concentrations and the eventual nucleation and slow growth of coarse crystalline CCC crystals and aggregates thereof.CCC formation requires conditions very close to 0 °C, as shown by clumped isotope data29. Mapping of CCC and detached stalagmites, however, only provides a minimum estimate of the former extent of ice in these caves. The presence of broken stalagmites and stalactites in other rooms suggests that the ice extent may have been larger, but these features could also be unrelated to ice deformation.CCC thus document that perennial ice close to the melting point was wide-spread in the Obir caves during the LGM. This is consistent with the occurrence of detached stalagmites in the same rooms and galleries, a deformation feature known from caves that were located in the permafrost zone during the last glacial period and where ice movement has caused wide-spread damage of older stalagmite generations30. The ages of different CCC types within individual heaps commonly differ by up to several hundred years. There is a relationship neither between age and the stable isotopic composition nor between petrographic types and age. Close inspection of the C and O isotope data show that individual CCC accumulations fall along parallel linear trends  or trapping of cold air . Perennial ice accumulations in modern ice caves in the Alps are associated with these negative thermal anomalies. The mean annual air temperature in the ice-bearing parts of these caves is typically 3–4 °C lower than the MAAT at the same elevation outsideThe Obir caves are rather exceptional in this context and several lines of evidence suggest that their former ice accumulations reflect permafrost rather than a local thermal anomaly caused by airflow. First, owing to their hypogene origin, these caves were fairly isolated from the outside atmosphere prior to their discovery, strongly impeding air exchange with the atmosphere. Second, had there been forced airflow in these caves during the past (an unlikely scenario), the negative thermal anomaly would have been restricted to the area behind the lower  entrance, which is inconsistent with the distribution of CCC  and lack thermal anomalies, thanks to their hypogene origin. This setting is key for drawing quantitative conclusions about the paleoclimate and past environments above the caves using the thermal and chronological information registered by CCC.Heat-flow modeling shows that the initial cooling of rock above the Obir caves is compatible with a stadial climate as suggested by proxy data. Essential for this subsurface cooling is the absence of a significant stable snowpack in winter, which leads to heat loss from the ground and a progressive deepening of the permafrost base. In the model, the Obir caves reach subzero temperatures within <50 yr of the onset of the cooling, depending on their depth, eventually approaching −4 °C after several hundred years Fig. . This sc34 , but is consistent with the CCC timing and in particular with the extended period during which the depth range of the Obir caves remained close to 0 °C. This long-term stability close to the freezing point across a significant depth gradient . An early numerical study in conjunction with paleoglaciological observations indicates up to ~150 m thick permafrost just outside the margin of LGM piedmont glacier lobes on the northern rim of the Alps42. And a regional compilation of permafrost features attributed to the LGM suggests that the southern limit of discontinuous permafrost straddled the southern rim of the Alps43.Because CCC record conditions very close to the freezing point, the oldest date represents a minimum age for ice accumulation in a given cave. Perennial ice and permafrost conditions therefore already prevailed in the Obir caves prior to 26.6 ± 0.2 ka. Data on the distribution of permafrost during and prior to the LGM in the peri-Alpine realm are generally consistent with this observation. A study of rock glaciers in the Julian Alps south of the Karawanks identified the lowest rock glaciers at 1076 m a.s.l.16). The following interval of low stalagmite oxygen isotope values, precisely constrained by 230Th ages, marks an interval of pronounced advection of southerly derived moisture to the Alps and matches the Obir CCC data both in terms of onset and duration . Our data support the hypothesis that the snow fell primarily before the main winter season, i.e., in autumn, in order to permit the buildup of a significantly thick insulating blanket against the chill of the long LGM winters. The timing of this hydroclimate switch is not only in excellent agreement with the Sieben Hengste record but also with less precise but spatially more comprehensive paleoglacier data sets from the Alps .We examined samples using a Keyence VHX-6000 digital microscope, and analyzed their stable C and O isotope composition. The latter was determined using a Gasbench II linked to a ThermoFisher Delta V Plus isotope ratio mass spectrometer. The results were calibrated against international standards and reported relative to the VPDB standard with a long-term precision of δ230Th dating was performed at Xi’an Jiaotong University, China, using a ThermoFisher Neptune plus multi-collector inductively coupled plasma mass spectrometer. The method is described in ref. 60. Standard chemistry procedures were used to separate U and Th. A triple-spike (229Th–233U–236U) isotope dilution method was used to correct instrumental fractionation and to determine U and Th isotopic ratios and concentrations60. Uncertainties in U and Th isotopic measurements were calculated offline at the 2σ level, including corrections for blanks, multiplier dark noise, abundance sensitivity, and contents of the same nuclides in the spike solution. 234U and 230Th decay constants of ref. 60 were used. Corrected 230Th ages assume an initial 230Th/232Th atomic ratio of (4.4 ± 2.2) × 10−6, and the value for material at secular equilibrium with the bulk earth 232Th/238U value of 3.8. The correction is small and within the 2σ uncertainty range  sufficient air exchange with the outside atmosphere to prevent the buildup of a geothermal gradient at the depth range of the caves (42–67 m). The cave system lacks streams and perennial water ingress points but shows evidence of seepage flow, leading to locally abundant dripstone formations. Although this very slow discharge likely only has a subordinate role in the heat exchange between the surface and the caves it is (and was during the LGM) very likely sufficient to eliminate the small contribution from the geothermal heat flow. We, therefore, modeled the thermal regime of the Obir caves without a geothermal heat-flow component. The thermal diffusivity of limestone ranges from about 1.1 × 10−6 m2/s to 1.2 × 10−6 m2/s at room temperature63. We used a value of 1.2 × 10−6 m2/s to account for some air-filled porosity in the Triassic limestone. The atmospheric temperature forcing was expressed by a sinusoidal seasonal cycle using the temperature of the warmest month (July) and of the coldest month (January) constrained by proxy data from published sources (We modeled the thermal conditions in the subsurface using a 1d heat-flow model, solving the heat equation utilizing finite differences as space discretizations alongside a forward Euler time-stepping scheme, stabilized with a diffusion-type Courant–Friedrichs–Lewy condition. The assumption of conductive heat transfer is justified given the fairly isolated nature of the Obir caves, which lack evidence of thermal anomalies common in caves showing forced ventilation or cold-trap behavior. The thermal regime of the shallow subsurface is governed by the interplay between the surface heat flow and the geothermal heat flow. The latter is comparably small (~50 mW/m sources . ModelinSupplementary InformationDescriptions of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2


Pharmacological inhibition of Syk by PRT060318 or PKC by GF109203X only minimally reduced OA-induced Syk S297 phosphorylation. PP2A inhibition by OA preceding GPVI-mediated platelet activation induced by convulxin extended Syk S297 phosphorylation but inhibited Syk Y-phosphorylation. Our data demonstrate a novel biochemical and functional link between the S-protein phosphatase PP2A and the Y-protein kinase Syk in human platelets, and suggest that PP2A represents a potential enhancer of GPVI-mediated Syk activity caused by Syk pS297 dephosphorylation.Distinct membrane receptors activate platelets by Src-family-kinase (SFK)-, immunoreceptor-tyrosine-based-activation-motif (ITAM)-dependent stimulation of spleen tyrosine kinase (Syk). Recently, we reported that platelet activation via glycoprotein (GP) VI or GPIbα stimulated the well-established Syk tyrosine (Y)-phosphorylation, but also stoichiometric, transient protein kinase C (PKC)-mediated Syk serine(S)297 phosphorylation in the regulatory interdomain-B, suggesting possible feedback inhibition. The transient nature of Syk S297 phosphorylation indicated the presence of an unknown Syk pS297 protein phosphatase. In this study, we hypothesize that the S-protein phosphatase 2A (PP2A) is responsible for Syk pS297 dephosphorylation, thereby affecting Syk Y-phosphorylation and activity in human washed platelets. Using immunoblotting, we show that specific inhibition of PP2A by okadaic acid (OA) alone leads to stoichiometric Syk S297 phosphorylation, as analyzed by Zn IIbβ3. This initial adhesion leads to platelet activation with multiple responses, including cytoskeletal remodeling, integrin αIIbβ3, activation, degranulation, synthesis/release of thromboxane A2 (TxA2), and surface exposure of anionic phospholipids. Functionally, this results in platelet shape change, enhanced adhesion, aggregation, and coagulant activity to form a thrombus [2+ concentration, and activate Ca2+-dependent protein kinases and protein kinase C (PKC), thereby inducing platelet activation [2) and nitric oxide (NO), decrease many of these platelet-activating pathways via the elevation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), respectively, and stimulation of their target protein kinases [Human platelets are small anucleate blood cells, which are essential components of the hemostasis system and widely recognized as circulating sentinels of the vessel wall ,2,3. Thethrombus ,6. Thesethrombus ,6,7, a sthrombus ,9. GPCR-tivation ,9. Endot kinases ,11. Whil kinases ,13, a si kinases ,15,16. G kinases ,18.IIbβ3 [Several members of the SFKs, such as Src, Lyn, Yes, Fyn, and feline Gardner-Rasheed sarcoma viral oncogene homolog (FGR), are highly expressed in human platelets and are important components of adhesion-dependent platelet activation mediated by GPVI, GPIb-V-IX, CLEC-2, and integrin αIIbβ3 ,6,7. A cIIbβ3 ,20,21. UIIbβ3 ,23,24. IIIbβ3 ,26. An iWhereas other major serine/threonine or tyrosine protein kinases such as PKC or SFKs have multiple family members expressed in human platelets, Syk has a crucial position, since it is the only member of its family expressed in human and murine platelets ,17. The Syk gene (by deletion of one exon of the Syk gene encoding for 41 residues in the Syk kinase domain in embryonic stem cells) die from severe hemorrhage before birth [Mice embryos presenting with a homozygous targeted mutation in the re birth . Mice lare birth , highligWhile the primary activation of Syk is tightly controlled by a few tyrosine-phosphorylation sites, there is increasing evidence, obtained mostly with murine and human B-cells, that Syk contains multiple additional tyrosine (Y), serine (S), and threonine (T) phosphorylation sites, which are thought to be important for recruiting additional regulatory proteins ,20,29. PRecently, we showed that specific activation of human platelets via GPVI (convulxin) or GPIbα (echicetin beads) not only stimulated transient Syk tyrosine phosphorylation and Syk activation, but also stoichiometric, transient, PKC-mediated phosphorylation of Syk S297 . MoreoveIn previous studies, we observed that activation of both GPIbα or GPVI caused a strong but transient stimulation of Syk S297 phosphorylation, suggesting activity of both a very active Syk S297 protein kinase but also protein phosphatase . In ordeWe recently described time-dependent convulxin (Cvx), GPVI-mediated effects on Syk-activation and S297 phosphorylation . TherefoPreviously, we showed by conventional SDS-PAGE plus phos-tag analysis that the transient, Cvx-induced phosphorylation of Syk S297 was stoichiometric and mediated by both Syk itself and PKC . To inveWe aimed to study the effect of PP2A inhibition on Cvx-induced Syk phosphorylation and Syk activity . PP2A inInterestingly, the inhibition of PP2A under these conditions showed only minimally inhibitory effects on the Cvx-stimulated phosphorylation of Syk Y352 and S297, which are both located in the interdomain-B of Syk c. HoweveWe aimed to extend our observations on the direct effect of OA on Syk S297 phosphorylation evoked by Cvx. Therefore, we pretreated washed platelets with vehicle control (EtOH) or with OA (1 µM) for 30 min, and then with Cvx (50 ng/mL). We previously showed that Cvx induces transient S297 phosphorylation. Our data here demonstrate that PP2A inhibition prolongs Syk S297 phosphorylation up to 30 min after Cvx stimulation , best seSamples from the same experiment shown in These results show that PP2A inhibition in human platelets differentially regulates Syk serine and tyrosine phosphorylation and reduces Cvx-stimulated Syk activity in human platelets.The Syk tyrosine protein kinase is essential for the activation and subsequent responses of platelets and immune cells. Syk is also known to be tightly regulated by multiple tyrosine and also serine/threonine protein kinases with their multiple phosphorylation sites. Very recently, we demonstrated and reported for the first time that GPVI-induced activation of human platelets caused a rapid, stoichiometric, transient phosphorylation of Syk S297, which is located at the crucial Syk interdomain-B . These a50~0.2 nM), directly binding to and inhibiting the PP2A catalytic subunit C, but other protein phosphatases only at much higher concentrations [Our present data show that incubation of intact human platelets with 1 µM okadaic acid (OA) caused the time-dependent appearance of Syk pS297, as detected by phosphosite-specific antibodies combined with regular western blot and phos-tag analyses a–c. Sevetrations ,35,36. Itrations  and the trations . Human ptrations . In agreThe combined analysis of Syk S297 phosphorylation by regular western blots and by phos-tag analysis c showed The phosphorylation state of a given protein phosphosite, here, Syk S297, in intact cells is determined by the equilibrium of the corresponding protein kinase and protein phosphatase activity using this site as the substrate. Although there is some basal protein phosphorylation in resting platelets as detected by phosphoproteomics , most reThe effects of PP2A inhibition on Syk S297 phosphorylation were then compared with the effects on Syk tyrosine phosphorylation and Syk activity by studying the Syk pY352 and pY525/526 phosphosites (as markers of Syk activation) and the tyrosine phosphorylation of the Syk substrates LAT (pY191) and PLCγ2 (pY759) as markers of Syk activity. We previously showed that PKC inhibition prevented GPIbα-/GPVI-induced Syk S297 phosphorylation but, in parallel, enhanced Syk Y352 and Y525/526, as well as LAT Y191 and PLCγ2 Y759 phosphorylation. This PKC-induced Syk S297 phosphorylation was proposed to be a mechanism of feedback inhibition . Our preOur results showed that PP2A inhibition differentially regulates Syk serine ↑ pS297) and tyrosine phosphorylation sites  and that PP2A inhibition reduces Cvx-stimulated Syk activity in human platelets. These effects are a mirror-image, at least in part, of our studies with pharmacological and physiological PKC inhibitors during Cvx-induced platelet activation, which were shown to abolish Syk S297 phosphorylation but enhance Syk tyrosine phosphorylation and activity . The pro7 and tyrClearly, there are limitations of these present studies. We did not identify the precise mechanism by which Syk S297 phosphorylation/dephosphorylation controls Syk activity. Future studies should analyze the precise molecular role of the Syk S297 phosphosite using structural, mutational, and further cellular/functional approaches, since it represents a regulatory hotspot for a tyrosine kinase crucial for the function of platelets and many immune cells. Since Syk is controlled by multiple post-translational modifications and protein interactions , it is pA remarkable result of our studies with human platelets is that Syk S297 phosphorylation shuttles between complete PP2A-catalyzed dephosphorylation and complete phosphorylation, induced by pathways activating PKC, Akt, and perhaps additional protein kinases  responsible for this activity. PP2A is not a single enzyme but a large protein family, with more than 92 different heterotrimeric forms present in humans ,46 and 1This study established an important interaction between the PP2A family and the tyrosine kinase Syk in human platelets, which is also likely to be significant regarding the regulation of immune cells and other tyrosine kinases, thereby requiring further investigation. This S297 site regulated by phosphorylation/dephosphorylation is located within the N-terminal Syk interdomain-B, whereas the well-established Y348/Y352 phosphosites important for activation are located within the C-terminal part of this linker domain. Overall, this Syk interdomain-B is an important molecular switch, which could allow graduated levels of Syk activity, depending on the state of interdomain and kinase domain phosphorylation. Such graduated activity of Syk was suggested to be important for tonic signaling, where complete or long-term Syk activation may not be desirable . Syk wasBlood was collected from 10 sex-matched, healthy volunteers (age 24–70 years) without taking platelet-affecting drugs and without presenting inflammatory, infectious, or allergic symptoms for at least 14 days, respectively, including normal blood counts. The health statuses of volunteers were routinely approved by the local medical office of the University Medical Center Main. All blood donors gave their informed consent. This study was approved in accordance with the Declaration of Helsinki by the local Ethics Committee of the University Medical Center Mainz .bis(β-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA) was centrifuged at 200× g for 10 min at room temperature (RT) to generate platelet-rich plasma (PRP). PRP was diluted 1:1 with citrate-glucose-sodium (CGS) washing buffer  and centrifuged at 69× g for 10 min at room temperature (RT) to pellet the leukocytes. The supernatant was centrifuged at 400× g for 10 min at RT to pellet the platelets. Each platelet pellet was resuspended in 3 mL CGS buffer and centrifuged at 400× g for 10 min at RT. Finally, platelets were resuspended in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer . The platelet count was adjusted to 5 × 108/mL and platelets were kept at 37 °C prior to treatment, which was also performed at 37 °C without stirring. A higher platelet count was used because the OA preincubation was already established with higher platelet counts and worked well for these conditions. For comparable results, all experiments needed to be done with the platelet count of 5 × 108/mL.Human platelets were washed and isolated as previously described ,34,38,53v/v) glycerol, 6% (w/v) SDS, 0.06% (w/v) bromphenol blue; 1:10 β-mercaptoethanol) in a ratio of 1:10. Samples were boiled at 95 °C for 10 min under gentle shaking. Platelet proteins were separated by electrophoresis using 8% SDS-polyacrylamide gels or Zn2+ Phos-tagtm-SDS-PAGE (6% (v/v) acrylamide, 35 µM Zn2+-phos-tag) followed by immunoblotting, as previously described [2+ Phos-tagtm-SDS-PAGE separated proteins according to their phosphorylation ratio, so only an antibody against the total protein was necessary. After transfer, polyvinylidene difluorid (PVDF)-membranes were blocked with 5% bovine serum albumin (BSA) in 20 mM tris, 140 mM NaCl, 0.1% (v/v) Tween®-20 , pH 7.4) for 1 h at RT and incubated overnight with the respective antibodies in 5% BSA 1xTBST at 4 °C. Membranes were washed three times with 1xTBST and incubated for 1 h at RT with the secondary-horseradish peroxidase (HRP)conjugated antibodies in 5% BSA–TBST. After three washes with 1xTBST, membranes were developed by electrochemiluminescence (ECL) detection. Phosphoantibodies against Syk S297, Y525/526, Y352, LAT Y191, PLCγ2 Y759, and β-actin  or anti-Syk, anti-PLCγ2  were used, as previously described [Washed platelets were prepared in one stock and separated for treatment. They were treated at 37 °C without stirring. Washed platelets were pre-incubated with 1 µM OA or EtOH (vehicle control), 20 µM tautomycetin for 10 min, or with 1 µM PRT or 5 µM GFX or DMSO (verhicle control) for 5 min at 37 °C. Pretreated samples were stimulated/inhibited with 50 ng/mL convulxin or 1 µM/2 µM OA or EtOH. Samples were taken after the corresponding time points and added to a 1 mL tube already containing 3× Laemmli buffer . Two-way ANOVA, followed by Sidak’s multiple comparison test for comparison of two groups at the same time point was used.


All oxindole–indole conjugates, except 6i, 9b, and 9c efficiently affected the growth of the human breast cancer MCF-7 (IC50: 0.39 ± 0.05–21.40 ± 1.58 μM) and/or MDA-MB-231 (IC50: 1.03 ± 0.04–22.54 ± 1.67 μM) cell lines, whereas bioisosteric replacement of the oxindole nucleus with indane or tetralin rings  diminished the anti-proliferative activity. In addition, hybrids 6e and 6f displayed effective cell cycle disturbance and proapoptotic capabilities in MCF-7 cells. Furthermore, the efficient anti-proliferative agents towards MCF-7 and/or MDA-MB-231 cell lines  were investigated for their potential inhibitory action toward CDK4. Hybrids 6a and 6e displayed good CDK4 inhibitory activity with IC50s equal 1.82 and 1.26 µM, respectively. The molecular docking study revealed that oxindole moiety is implicated in two H-bonding interactions via both (NH) and (C=O) groups with the key amino acids Glu94 and Val96, respectively, whereas the indole framework is stably accommodated in a hydrophobic sub-pocket establishing hydrophobic interactions with the amino acid residues of Ile12, Val20, and Gln98 lining this sub-pocket. Collectively, these results highlighted hybrids 6a and 6e as good leads for further optimization as promising antitumor drugs toward breast malignancy and CDK inhibitors. On account of their overexpression in a wide range of human malignancies, cyclin-dependent kinases (CDKs) are among the most validated cancer targets, and their inhibition has been featured as a valuable strategy for anticancer drug discovery. In this study, a hybrid pharmacophore approach was adopted to develop two series of oxindole–indole conjugates ( Cancer is a serious health problem that is considered a leading cause of death worldwide. Cancer is characterized by uncontrolled cell proliferation and differentiation mechanisms . AlthougCyclin-dependent kinases (CDKs) are categorized as protein serine/threonine kinases that are responsible for regulating the cell cycle through the complexation with regulatory proteins known as “cyclins”. These complexes are responsible for the progression of the cell cycle through its different phases ,6. This CDK4 is a key CDK cell cycle regulator that is responsible for the progression from the G1 to S phase through complexation with cyclin-D at the G1-phase ,12. Seve®, β (GSK-3β) [As a privileged scaffold, isatin  represents a leading and promising heterocyclic nucleus that is endowed with a range of interesting biological properties, chiefly antitumor activities . Upon FD(GSK-3β) , aurora (GSK-3β) , p90 rib(GSK-3β) , and mic(GSK-3β) , to nameI–III [In the current medical era, the molecular hybridization strategy is being featured as a promising drug design approach, particularly in anticancer drug discovery ,30. It iI–III ,33,34, FI–III ,36, inhiI–III ,38, in aI–III ,40.6a–i, 9a–f, and 11a,b) aiming to develop efficient antitumor agents with potential inhibitory action toward CDK4. First, the privileged indole moiety was selected to be conjugated with the isatin scaffold to furnish the first series (6a–i); thereafter, N-1 of the oxindole moiety was alkylated and benzylated to produce the second set of target hybrids (9a–f)  with a view to investigating the significance of the oxindole moiety in achieving the desired biological activities  . Finallytivities . 6a–i, 9a–f, and 11a,b) were examined for their potential cytotoxic activity towards MCF-7 and MDA-MB-231 cancer cell lines via SRB assay. Thereafter, hybrids 6e and 6f were further explored for their plausible influence on cell cycle progression and apoptosis induction possibility in breast cancer MCF-7 cells. In addition, the most efficient anti-proliferative agents towards MCF-7 and/or MDA-MB-231 cells  were examined for their inhibitory action toward CDK4, and then docked in the CDK4 binding site to justify their inhibitory action as well as to explore their binding pattern.All the reported conjugates  in boiling ethanol to produce the key intermediate 1H-indole-2-carbohydrazide 4. Final target hybrids 6a–i were obtained via condensation of 1H-indole-2-carbohydrazide 4 with the appropriate unsubstituted (5a), 5-substituted (5b–h), and 5,7-disubstituted (5i) isatin derivative in refluxing glacial acetic acid for four hours  were examined for the potential cytotoxic activity towards breast cancer MCF-7 and MDA-MB-231 cell lines, utilizing the protocol of the sulforhodamine B colorimetric (SRB) assay [6, 9, and 11 were designed with diverse substituents on C-5 and C-7 of the oxindole moiety; R and R1 in series 6a–i, and diverse substitutions on the N-1 of the oxindole moiety; and R2 in series 9a–f, while the oxindole moiety was replaced by an indane and tetralin rings in compounds 11a,b in order to explore the privilege of the oxindole moiety over other pharmacophoric groups. The IC50 values for the tested conjugates were compared as an insight of their anti-proliferative potency taking the clinically approved staurosporine as the positive control anticancer drug (All the synthesized conjugates (B) assay . With thcer drug .50 values for the tested hybrids toward MCF-7 breast cancer cells revealed that most hybrids belonging to series 6 had excellent activity against the aforementioned cell line with IC50 values spanning between 0.39 ± 0.05 μM and 6.00 ± 0.32 μM, with the exception of compounds 6h and 6i  bearing a strong electron withdrawing 5-NO2 group and 5,7-dimethyl substitution on the oxindole moiety, respectively  revealed that it had double the activity of staurosporine (IC50 = 6.81 ± 0.22 μM), which highlighted that this scaffold is a promising one. The influence of grafting different groups on the oxindole moiety reflected that substitution with a halide was advantageous for the anti-proliferative activity in case of 5-chloro substituent in 6c, as it displayed slight potency enhancement compared to its unsubstituted analogue 6a , while the 5-bromo derivative 6d (IC50 = 3.31 ± 0.11 μM) did not prove to be more effective than the unsubstituted derivative 6a (IC50 = 3.12 ± 0.14 μM), and even the 5-fluoro derivative 6b (IC50 = 6.00 ± 0.32 μM) showed about half the activity in comparison to 6a (IC50 = 3.12 ± 0.14 μM).The IC6e (IC50 = 0.39 ± 0.05 μM) markedly boosted the activity 8-fold compared to the unsubstituted prototype 6a (IC50 = 3.12 ± 0.14 μM), emphasizing that an electron donating group is advantageous for activity. In accordance with this finding, compound 6f, bearing an electron-donating 5-OCH3 group, also exhibited excellent anti-proliferative activity (IC50 = 1.85 ± 0.08 μM) with 1.7-fold efficacy enhancement as compared to 6a (IC50 = 3.12 ± 0.14 μM). Unfortunately, compound 6g, which possesses a trifluoromethoxy group, did not exhibit superior anti-proliferative activity compared to 6a, yet it still proved to be more efficient than the reference compound, staurosporine, with an IC50 value of 5.07 ± 0.21 μM.Furthermore, C-5 monosubstitution of the oxindole motif with a methyl group in 9 did not show better cytotoxic action against the MCF-7 cell line than their N-unsubstituted analogues 6a–i;  and , indicating that both N-alkylation and N-benzylation for the oxindole nucleus is not advantageous for the growth inhibitory action toward MCF-7 breast cancer cell line. N-1 substitution of the oxindole moiety with a methyl group produced compound 9a, which is slightly more potent toward the MCF-7 cell line compared to the reference staurosporine . Alternatively, compound 9b, bearing an n-propyl group, and 9c, possessing an ester group, showed very poor anti-proliferative action against MCF-7 cancer cells with IC50s equal to 40.89 ± 3.17 μM and 67.51 ± 3.02 μM, respectively. While compound 9d, bearing a benzyl ring, showed moderate anti-proliferative activity with an IC50 value of 9.56 ± 0.61 μM, compound 9e, with a fluorobenzyl group, proved to be three-fold more potent compared to its counterpart 9d with an unsubstituted benzyl group with an IC50 of 3.70 ± 0.19 μM, On the other hand, the tested hybrids of series 9f, bearing an n-propyl group, displayed about two-fold enhanced activity (IC50 = 19.26 ± 0.65 μM) toward the MCF-7 cell line than its C-5 unsubstituted counterpart 9b (IC50 = 40.89 ± 3.17 μM), highlighting that C-5 substitution may be beneficial for the growth inhibitory activity of the N-substituted oxindole-based hybrids against MCF-7 cells. Moreover, it indicates that a chloro substitution at the oxindole C-5 is beneficial for activity, as also seen in compound 6c.Moreover, the 5-chlorooxindole-based hybrid 50 profile for the tested hybrids against the MDA-MB-231 cell line unravels very interesting results. All the compounds showed a narrower range of IC50 values against MDA-MB-231 as compared to their corresponding values toward the MCF-7 cell line. The IC50s were ranging from 1.03 ± 0.04 μM to 22.54 ± 1.67 μM, which proves that the examined conjugates exhibited more potent growth inhibitory action against TNBC, MDA-MB-231, with the exception of conjugates 6i, 9c, 11a, and 11b, which showed no activity toward the tested cell line. Regarding the IC50 values of series 6a–i, the unsubstituted derivative, 6a, did not show superior activity to staurosporine , whereas C-5 substitution of the oxindole nucleus with a halide was suggested to be advantageous for the anti-proliferative action regarding the fluoro substitution in 6b (IC50 = 6.36 ± 0.29 μM), which is more potent than the reference compound (IC50 = 10.29 ± 0.72 μM). Moreover, grafting a chloro group afforded 6c with comparable potency to staurosporine , while substitution with a bromo group afforded 6d (IC50 = 18.33 ± 0.71 μM), which is much less potent than staurosporine. This proves that increasing the size of the halide group is detrimental for the cytotoxic activity against MDA-MB-231. Unexpectedly, grafting a methyl group was not advantageous for the growth inhibitory action toward breast cancer MDA-MB-231 cells in contrast to activity recorded against MCF-7, as the IC50 of 6e against MDA-MB-231 was 22.54 ± 1.67 μM, which is almost half the potency of staurosporine. Fortunately, grafting a stronger electron donating group, a methoxy group, boosted the activity of 6f (IC50 = 2.85 ± 0.9 μM) 3.6-fold as compared to staurosporine (IC50 = 10.29 ± 0.72 μM). In addition, substitution with a trifluoromethoxy group imparted 6g, which is more potent than the reference drug . Surprisingly, grafting a strong electron withdrawing group, a nitro group, afforded 6h, which is 5.56-fold more potent compared to staurosporine . Increasing the alkyl group to a propyl group afforded 9b which has a greater IC50 value (18.25 ± 0.85 μM), suggesting that increasing the bulkiness of the alkyl group is unfavorable for the anti-proliferative activity. Conversely, substitution with a benzyl group afforded 9d, which exhibited good anti-proliferative activity in comparison to staurosporine . Replacement of the benzyl ring with a fluorobenzyl group even boosted the activity ten-fold as compared to staurosporine .Regarding the antiproliferative action of series 11a,b), which did not exert any significant anti-proliferative action toward MCF-7 and MDA-MB-231 cancer cell lines  and the anti-apoptotic marker Bcl-2 was investigated to prove that our tested compounds exert their antiproliferative activity by driving the cell to apoptosis. Auspiciously, both conjugates evidently enhanced the level of the proapoptotic Bax protein by 8.5- and 7-fold, respectively. Homogeneously, both conjugates were able to decrease the level of the anti-apoptotic Bcl-2 protein by 2.12-, and 1.7-fold, respectively  were initially evaluated for their percent inhibition of CDK4 at a dose of 20 μM (6a (92%) and 6e (93%) showed stunning inhibitory activities, the rest of the examined hybrids showed low to moderate activity with percent inhibition spanning in the range of 12% to 46%   . The presented results in 6a and 6d were the best developed CDK2 inhibitors here with percent inhibition of 78% and 85%, respectively. In addition, compounds 6c, 6e, 6f, and 6h displayed percent inhibition equal to or higher than 50% toward this isoform at a concentration of 20 μM.On the other hand, inhibition of CDK2 ranged from good to weak, with percent inhibition range of 45%–85% . In partIn conclusion, the results of inhibition of the three tested CDK isoforms  disclosed the inability of the target compounds to inhibit CDK9 significantly, with concomitant good inhibitory activities against CDK2 and CDK4. Accordingly, further optimization for the here reported oxindoles considering their plausible dual CDK2 and CDK4 inhibitory actions are currently in development and expected to be conveyed in the future.A protein data bank (PDB) search showed that there are not any CDK4 protein structures co-crystalized with an inhibitor available (holoprotein). The available structures are apoprotein structures for CDK4 in complex with cyclin D such as PDB ID 2W96, 2W9Z, 2W9F, and 2W99 . On the First, the molecular docking protocol was validated by performing self-docking for the added oxindole ligand in the vicinity of the CDK4 binding site. The self-docking validation replicated the binding pattern for the added ligand, efficiently demonstrating the aptness of the used setup for the planned docking study, and this was confirmed by the low RMSD of 0.879 Å between the docked pose and the added ligand (energy score (S) = −9.72 kcal/mol) .6a–i showing promising cytotoxic as well as CDK4 inhibitory activity were docked in the CDK4 binding site in order to justify their inhibitory action and investigate their binding patterns. In general, hybrids 6a–i achieved similar binding patterns, where the oxindole framework was fitted in the ATP adenine binding pocket like that of the added ligand in the hinge region. As observed in 9 with N-substituted derivatives. Moreover, the oxindole ring interacts by its phenyl part through π–π stacking interaction with the side chain phenyl of Phe93 and through hydrophobic interaction with the side chains of Phe93 and Ala157. A hydrophobic substituent with the appropriate size on the oxindole ring 5-position could improve the binding affinity, which rationalizes the promising activity of 6e with a 5-methyl substitution. Thus, derivatives in this series carrying substituents with hydrophilic characters, 6b, 6f, and 6g, or bulky size, 6c, 6d, 6h, and 6i, showed lower CDK4 inhibitory activity as reflected in their CDK4 inhibitory actions and docking scores . All coupling constant (J) values were expressed in hertz. The used abbreviations are as following: s, singlet; d, doublet; m, multiplet. Compound 3 [8a–f [4 [6a–d [Infrared (IR) spectra were recorded as KBr disks by the use of a Schimadzu FT-IR 8400S spectrophotometer. NMR spectra were recorded on JEOL ECA-500 II (500/125 MHz) or Bruker (400/100 MHz) NMR spectrophotometers using deuterated dimethylsulfoxide  in ethyl alcohol (25 mL). The resulting reaction mixture was heated under reflux for two hours. Thereafter, it was cooled to r.t. and then poured over crushed ice. The formed solid was filtered-off, washed with water (3 × 5 mL), and recrystallized from isopropyl alcohol to produce the key intermediate 1H-indole-2-carbohydrazide 4. An excess of 99% hydrazine monohydrate  was added to a hot solution of ethyl indole-2-carboxylate 4  in glacial acetic acid (15 mL) was treated with the appropriate isatin derivative 5a–i and 8a–f (1.2 mmol), or ketones 10a and 10b (1.2 mmol). The resulting reaction mixture was refluxed for four hours then cooled to r.t. The obtained precipitate was collected by filtration and dried to get a powder that was recrystallized from glacial acetic acid to furnish the titled conjugates 6a–i, 9a–f, and 11a,b.A solution of indole-2-carbohydrazide 4) and the target hybrids  are provided in the Details for the full characterization of the key intermediate  are provided in the Details for experimental protocols used in the diverse biological assays for the targeted hybrids  that obtained from American Type Culture Collection. Cytotoxicity was assessed following the SRB colorimetric assay protocol [Target compounds protocol , as repoprotocol ,51.6e and 6f at their IC50 , by BD FACS Calibur flow cytometer, as described previously [Flow cytometric analysis (FACS) was performed to assess the cell cycle distributions in MCF-7 breast cancer cells after treatment by hybrids eviously .6e and 6f towards MCF-7 cells was evaluated through an FITC Annexin V apoptosis detection kit by flow cytometry, in accordance with the manufacturer’s protocol and referring to the reported procedures [Assessment of the levels for the proapoptotic markers  as well as the antiapoptotic marker (Bcl-2) was carried out through the use of ELISA colorimetric kits in accordance with the manufacturer’s instructions, as mentioned earlier . In addiocedures .http://www.reactionbiology.com) Kinase HotSpotSM service.The in vitro CDK kinase inhibition assay was performed by Reaction Biology Corp.  (MatchMaker command available in UCSF Chimera package 1.12 [The CDK2 protein structure (PDB ID: 4FX3) co-crystalized with an oxindole inhibitor and the CDK4 protein structure (PDB ID: 2W96)  were dowage 1.12 . Then, tFor carrying out the molecular docking for the target hybrids in the CDK4 binding site, the following procedures were adopted. Pose mode [(A) Ligand preparation: The synthesized hybrids as well as the added oxindole ligand were built as 3D structures using Discovery Studio Visualizer 2017R2 . The OMEose mode ,56.(B) Protein preparation: The generated structure for CDK4 in step 3.2.5.1. with the added oxindole inhibitor was used in this step. Using Discovery Studio Visualizer 2017R2 , the proChemgauss4 scoring function [(C) Molecular docking: The HYBRID docking module of “OEDocking 3.2.0.2” program in OpenEye package was utilized to carry out the molecular docking for the generated conformers of the herein reported hybrids as well as the oxindole inhibitor in the CDK4 binding site using the function ,58,59,60First, the molecular docking protocol was validated by performing self-docking for the added oxindole ligand in the CDK4 binding site, producing a docking pose with energy score (S) and RMSD equal to −9.72 kcal/mol and 0.879Å, respectively. Then, this validated docking setup was adopted to investigate the ligand–target interactions in the CDK4 active site for the prepared hybrids to explore their binding pattern and to justify their binding affinity. PoseView 1.1.2 was used to generate the 2D figures of the ligand–target interactions ,62,63,646a–i and 9a–f) and carbocycle–indole hybrids  as efficient antitumor agents with potential inhibitory action toward CDK4. All the prepared hybrids  were examined for the potential cytotoxic activity towards MCF-7 and TNBC MDA-MB-231 breast cancer cell lines via SRB assay. The synthesized oxindole–indole conjugates, except 6i, 9b and 9c, efficiently affected the growth of the human breast cancer MCF-7 (IC50: 0.39 ± 0.05–21.40 ± 1.58) and/or MDA-MB-231 (IC50: 1.03 ± 0.04–22.54 ± 1.67) cell lines, whereas bioisosteric replacement of the oxindole motif with indane or tetralin rings  diminished the anti-proliferative activity, indicating the importance of the oxindole scaffold for the antitumor activity. Moreover, hybrids 6e and 6f triggered cell cycle arrest and apoptosis in MCF-7 cancer cells as explicated by their capabilities to considerably boost the Bax/Bcl-2 ratio, and to up-regulate the level of caspase-3 and p53. Furthermore, single dose (20 μM) inhibitory activity of the most efficient anti-proliferative agents  was initially evaluated toward CDK4. Hybrids 6a and 6e showed the most potent inhibitory activity of 92% and 93%, respectively, whereas the rest of the examined hybrids showed low to moderate activity with percent inhibition of 12%–46%. Thereafter, the IC50 values for hybrids 6a and 6e in CDK4 were further determined and were found to be 1.82 and 1.26 µM, respectively. The molecular docking study revealed that the oxindole moiety was engaged in two hydrogen bonding interactions via both (NH) and (C=O) functionalities with the key amino acids Glu94 and Val96, respectively, rationalizing the lower activity of series 9 with N-substituted derivatives. The oxindole ring interacted by its phenyl part through π–π stacking interactions with the side chain phenyl of Phe93 and through hydrophobic interaction with the hydrophobic side chains of Phe93 and Ala157. In addition, the indole framework was stably accommodated in a hydrophobic sub-pocket, establishing a hydrophobic interaction with the hydrophobic side chains of amino acids Ile12, Val20, and Gln98 lining this sub-pocket. Finally, compounds 6a–h, 9a, and 9e were screened for their potential inhibitory activities against CDK2 and CDK9 isoforms. While CDK9 was weakly inhibited with percent inhibition range of 4%–32%, inhibition of CDK2 ranged from good to weak, with percent inhibition range of 45%–85%. Collectively, these results highlighted hybrids 6a and 6e as good leads for further optimization as promising antitumor drugs toward breast malignancy and CDK inhibitors.The current study presents the development of two series of oxindole–indole hybrids (


The need for biochemical characterisation of each putative LPMO is discussed based on recent reports showing that not all proteins with a Cu-histidine brace are enzymes.Lytic polysaccharide monooxygenases (LPMOs) are mononuclear copper enzymes that catalyse the oxidative cleavage of glycosidic bonds. They are characterised by two histidine residues that coordinate copper in a configuration termed the Cu-histidine brace. Although first identified in bacteria and fungi, LPMOs have since been found in all biological kingdoms. LPMOs are now included in commercial enzyme cocktails used in industrial biorefineries. This has led to increased process yield due to the synergistic action of LPMOs with glycoside hydrolases. However, the introduction of LPMOs makes control of the enzymatic step in industrial stirred-tank reactors more challenging, and the operational stability of the enzymes is reduced. It is clear that much is still to be learned about the interaction between LPMOs and their complex natural and industrial environments, and fundamental scientific studies are required towards this end. Several atomic-resolution structures have been solved providing detailed information on the Cu-coordination sphere and the interaction with the polysaccharide substrate. However, the molecular mechanisms of LPMOs are still the subject of intense investigation; the key question being how the proteinaceous environment controls the copper cofactor towards the activation of the O-O bond in O The replacement of liquid fossil transportation fuels with ethanol produced from agricultural and forest lignocellulosic residues is now technically possible . HoweverLignocellulose is a complex recalcitrant matrix dominated by lignin, cellulose and hemicellulose, which provides the plant tissue with strength and durability. The most abundant polysaccharide in lignocellulose is cellulose, and several enzymes are involved in its decomposition into glucose. The process requires the synergistic action of endo-acting glycosyl hydrolases (endoglucanase), processive exo-hydrolases acting from the reducing end (cellobiohydrolase I), and processive exo-hydrolases acting from the non-reducing end (cellobiohydrolase II) of the cellulose chains. The overall reaction is driven towards completion by β-glycosidases through the alleviation of cellobiose product inhibition of the exo-enzymes . DespitePycnoporus coccineus (now Trametes coccineus) . Al. AlTaAA9ningitis . Intriguc TaAA9A . Only Taellulose  as was eAnother Cu-histidine brace found in nature is the B-site in particulate methane monooxygenases. These enzymes catalyse the oxidation of methane to methanol. Until recently, this site was identified as the active site, in part due to its similarities to the LPMO histidine brace. However, another mononuclear copper site with different coordination geometry more recently has been suggested to be the active site ,74. It iWithin the field of industrial biotechnology, LPMOs are of the utmost importance for efficient decomposition and processing of plant materials.LPMOs and LPMO-like proteins are widely found in nature. The Cu-active site of LPMOs undergoes redox-cycling to catalyse cleavage of polysaccharides using molecular mechanisms that rely on redox partners and use oxygen species as co-substrate.in vivo.Both enzymatic and abiotic oxidative processes must be carefully controlled in biorefineries. Further studies are required to shed further light on how LPMOs acts


It consists of a kinase cascade, in which MST1/2 and MAP4Ks phosphorylate and activate LATS1/2, which in turn phosphorylate and inhibit YAP/TAZ activity. A variety of signals can modulate LATS1/2 kinase activity to regulate Hippo pathway. However, the full mechanistic details of kinase-mediated regulation of Hippo pathway signaling remain elusive. Here, we report that TNF activates LATS1/2 and inhibits YAP/TAZ activity through MEKK2/3. Furthermore, MEKK2/3 act in parallel to MST1/2 and MAP4Ks to regulate LATS1/2 and YAP/TAZ in response to various signals, such as serum and actin dynamics. Mechanistically, we show that MEKK2/3 interact with LATS1/2 and YAP/TAZ and phosphorylate them. In addition, Striatin-interacting phosphatase and kinase (STRIPAK) complex associates with MEKK3 via CCM2 and CCM3 to inactivate MEKK3 kinase activity. Upstream signals of Hippo pathway trigger the dissociation of MEKK3 from STRIPAK complex to release MEKK3 activity. Our work has uncovered a previous unrecognized regulation of Hippo pathway via MEKK2/3 and provides new insights into molecular mechanisms for the interplay between Hippo-YAP and NF-κB signaling and the pathogenesis of cerebral cavernous malformations.The Hippo pathway is an evolutionarily conserved signaling pathway that controls organ size in animals  The Hippo signaling pathway plays various roles in the regulation of cellular behavior, organ size, and tissue homeostasis . DysreguThe Hippo pathway can respond to a variety of intrinsic or extrinsic signals, such as cell–cell contact, cell adhesion, cell polarity, matrix stiffness, mechanical stress, shear stress, energy stress, osmotic stress, and hormonal signals, such as lysophosphatidic acid (LPA) in the serum, which result in the alteration of LATS1/2 phosphorylation and kinase activity , 16, 17.MAP3K2) and MEKK3 (encoded by MAP3K3) as the kinases responsible for the TNF-induced LATS activation. MEKK2/3 are serine/threonine kinases belonging to the MAP3K family. MEKK2/3 are capable of activating multiple downstream MAPKs, including ERK1/2, JNK, MAPK/ERK5, and p38 , NIH3T3 , and lung cancer cell A549 (A). The phosphorylation of NF-κB subunit p65 validated the successful activation of NF-κB signaling in the TNF-treated HEK293A and NIH3T3 cells (A and B). The phosphorylation of LATS1/2 and YAP-S127 (a LATS phosphorylation site) was increased in HEK293A, NIH3T3, and A549 cells . The phosphorylation of MOB1 Threonine 35 (MOB1-T35) by MST1/2 was unchanged (A and B). YAP/TAZ protein levels decreased in conjunction with increased LATS1/2 and YAP phosphorylation (A and B), which suggests that TNF elicits LATS activation and subsequent YAP/TAZ inactivation independent of MST1/2. MAP4K family members (MAP4K1/2/3 and MAP4K4/6/7) and MST1/2 have been demonstrated to act direct LATS-activating kinases. The deletion of MAP4Ks and MST1/2 eliminates most of LATS-targeted YAP phosphorylation induced by various signals. Surprisingly, TNF still induced robust LATS phosphorylation and YAP/TAZ degradation in MM 8KO (combined deletion of MST1/2 and MAP4K1/2/3/4/6/7) HEK293A cells , indicating the existence of additional LATS-activating kinases to mediate TNF-induced LATS activation. To test the dependency of LATS phosphorylation on TNF/NF-κB signaling, we generated RIPK1 KO HEK293A cells using CRISPR/Cas9 technology. It has been shown that Ripk1−/− MEF cells fail to activate NF-κB in response to TNF stimulation . Interestingly, the deletion of RIPK1 blocked TNF-induced phosphorylation of LATS and YAP . Previously, we showed that TAK1 is involved in TNF-induced inactivation of YAP independent LATS1/2. TAK1 functions as a pivotal activator to mediate the activation of NF-κB . Furthermore, serum starvation and actin depolymerization stimulated LATS and YAP phosphorylation in Tak1 KO NIH3T3 cells similar as in control cells (C and D). These results suggest that TNF stimulates LATS kinase activity and the putative LATS-activating kinases may act downstream RIPK1 and independent of TAK1.We previously found that TNF stimulates LATS kinase activation in primary chondrocytes . To furtFig. 1ell A549 A. The phmulation . Indeed,of NF-κB , 40. HowA). In contrast, other MAP3K family members exhibited no significant kinase activities on LATS1 (A and B). Furthermore kinase-dead forms of MEKK2 (MEKK2-KD) and MEKK3 (MEKK3-KD) failed to increase LATS1 phosphorylation (A). We next tested whether MEKK2/3 could promote MST1/2 kinase activity and indirectly activate LATS kinase activity. The overexpression of MEKK2 and MEKK3 had no kinase activity on MST1/2 shown by MST1 phosphorylation (C) and MOB1-T35 phosphorylation (D), a known MST1/2-specific phosphorylation site. Importantly, MEKK2 and MEKK3 still promoted LATS1 phosphorylation in MM 8KO HEK293A cells in a kinase-activity-dependent manner (B). Moreover, MEKK3 promoted YAP-S127 phosphorylation in MM 8KO HEK293A cells, but not in LATS1/2 DKO HEK293A cells (C), thereby further confirming that MST1/2 and MAP4Ks are not required for MEKK2/3 to induce LATS and YAP phosphorylation. In addition, we found that MAP4K4 and MST1 efficiently induced LATS and YAP-S127 phosphorylation in MEKK2/3 DKO HEK293A cells . Together, these results indicate that MEKK2/3 elicit LATS kinase activity independent of MST1/2 and MAP4Ks.To investigate how LATS kinases were activated by TNF, we screened the kinases related with NF-κB pathway to identify candidates that could induce the activation of LATS by coexpressing of candidate kinases with full-length Flag-tagged LATS1 and then examining the phosphorylation status of LATS1-T1079. MST1 was used as a positive control. By this approach, we identified MEKK2 and MEKK3, two highly homologous MAP3Ks (mitogen-activated protein kinase kinase) among 19 MAP3K super family that can efficiently promote LATS1 activation similar as MST1 A. In conon LATS1 , A and Brylation A. We nexrylation C and MOBrylation D, a knowt manner B. Moreov3A cells C, therebF and G and Fig. S2F). Furthermore, the interactions between MEKK2/3 and LATS1/2 were not mediated by YAP and TAZ, since MEKK2/3 still bound to LATS1/2 in YAP/TAZ knockdown cells (G–I). In addition, MEKK2 and MEKK3, but not their kinase-dead forms, phosphorylated LATS1 in the in vitro kinase assay . Together, our data suggest that MEKK2/3 act parallel to MST1/2 and MAP4K family kinases to activate LATS1/2 and phosphorylation of YAP in the Hippo pathway.Since MEKK2/3 promote LATS activation, we tested whether MEKK2/3 are able to interact with LATS kinases. As expected, we observed that MEKK2 and MEKK3 interacted with LATS1/2 at both exogenous and endogenous levels (LATS1/2 DKO HEK293A cells. YAP and TAZ interacted with MEKK2 and MEKK3 at both exogenous and endogenous levels (A–C and A). Furthermore, the deletion of WW domain in YAP abolished the interaction between MEKK3 and YAP (B). These results indicate that MEKK2/3 interact with YAP/TAZ independent of LATS1/2.It has been reported that human MEKK3 contains a PPXY motif (aa 178–181) providing a binding site for the WW domain of YAP . We foun A–C and A. Furthe and YAP B. These C). To further explore how MEKK2/3 contribute to YAP degradation, we examined whether MEKK2/3 promote YAP ubiquitination. We found that the expression of MEKK3 markedly enhanced poly-ubiquitination modification of YAP protein (D). Of note, MEKK3-induced YAP poly-ubiquitination was abolished in the absence of LATS1/2 (D). Conversely, YAP poly-ubiquitination was greatly reduced in MEKK2/3 DKO HEK293A cells (D and E). Furthermore, TNF-induced YAP poly-ubiquitination was abrogated in MEKK2/3 DKO HEK293A cells (E). Previous study demonstrates that LATS-induced YAP degradation is through SCF-β-TRCP-mediated ubiquitination (F). Consistent with the inhibitory function of MEKK2/3 on YAP, the expression of YAP target genes CTGF and CYR61 was decreased in MEKK2 or MEKK3 expressing HEK293A cells, but not their kinase-dead forms (G). Furthermore, the expression levels of CTGF and CYR61 were elevated in MEKK2/3 DKO HEK293A cells (H). In addition, we examined the effect of MEKK2/3 on YAP protein localization by immunofluorescence staining. MEKK2/3, but not their kinase-dead mutants, induced endogenous YAP to translocate into cytoplasm in HEK293A cells (I), indicating that this process requires MEKK2/3's kinase activity. Nuclear/cytosolic fractionation further supported the notion that MEKK3 promoted YAP cytoplasmic translocation (F). Importantly, MEKK3 still elicited YAP cytoplasmic translocation in MM 8KO, but not in LATS1/2 DKO HEK293A cells (G and H), which is consistent with the observation that MEKK3 promoted YAP-S127 phosphorylation through LATS1/2 (C). In addition, MAP4K4 stimulated YAP cytoplasmic translocation in MEKK2/3 DKO HEK293A cells (I). Thus, these results indicate that MEKK2/3 promote YAP proteasome-mediated degradation and cytoplasm translocation through LATS1/2.Phosphorylation of YAP/TAZ by LATS1/2 leads to YAP/TAZ cytoplasmic translocation and proteasome-mediated protein degradation. Next, we tested whether MEKK2/3 promote YAP degradation through LATS1/2. MEKK3 expression induced the degradation of endogenous YAP and TAZ in a dose-dependent manner in HEK293T cells, which can be blocked by MG132 administration C. To fur protein D. Of not LATS1/2 D. Conver3A cells , D and E3A cells E. Previotination . Notablytination F. Consisad forms G. Furthe3A cells H. In add3A cells I, indicalocation F. Import3A cells , G and H LATS1/2 C. In add3A cells I. Thus, A). The mobility shift of YAP was abolished when treated with λPPase (Lambda Protein Phosphatase) (A), indicating that YAP is phosphorylated. MEKK2/3 could activate LATS activity, which may result in the observed phosphorylation of YAP. To rule out this possibility, YAP-5SA, in which all LATS1/2 kinases phosphorylation sites were mutated, was used to coexpress with MEKK2 or MEKK3 in HEK293A cells. However, a remarkable mobility shift of YAP-5SA on SDS-PAGE was still induced in the presence of MEKK2 or MEKK3 (B). In addition, MEKK2/3 purified from LATS1/2 DKO HEK293A cells phosphorylated GST-YAP purified from E. coli in the in vitro kinase assay (B). To identify which amino acids in YAP were phosphorylated by MEKK2/3, we performed mass spectrometric analysis using YAP-Flag proteins purified from LATS1/2 DKO HEK293A cells coexpressing with either MEKK3-WT or MEKK3-KD. Mass spectrometry showed that phosphorylation of YAP at Ser371 and Thr412 was increased by MEKK3-WT but not its kinase-dead mutant (C). Next, we generated YAP mutations targeting Ser371 and Thr412 sites. Mutating the two sites largely eliminated MEKK3-induced YAP phosphorylation in the in vitro kinase assay (C) and the mobility shift of YAP in LATS1/2 DKO HEK293A cells on SDS-PAGE (D). To further investigate YAP phosphorylation by MEKK2/3, we generated a rabbit polyclonal antibody that specifically recognizes phosphorylated Ser371 of YAP (p-YAP-S371). This antibody recognized GST-YAP recombinant protein incubated with MEKK3-Flag but not MEKK3-KD-Flag immunoprecipitated from HEK293A cells in the in vitro kinase assay (D), indicating that MEKK3 indeed phosphorylates YAP at Ser371. Furthermore, we were able to detect exogenous and endogenous YAP phosphorylation at S371 by this antibody in MEKK3 overexpressing HEK293T cells (E and F). These data indicate that MEKK2/3 directly phosphorylate YAP.We always observed a mobility shift of YAP in SDS-PAGE when YAP was coexpressed with MEKK2 or MEKK3 A. The mophatase) A, indicaor MEKK3 B. In addse assay B. To ided mutant C. Next, se assay C and theSDS-PAGE D. To furse assay D, indica3T cells , E and FLATS1/2 DKO cells, respectively . Notably, the mutations of Ser371 and Thr412 to Ala in YAP largely abolished MEKK3-induced suppression on YAP/TEAD transcriptional activity in a Gal4/TEAD4-luciferase reporter assay . Furthermore, S371/T412 phosphomimetic form of YAP was phosphorylated at S127 by LATS1 similar as WT YAP , indicating that the phosphorylation of YAP by MEKK2/3 did not impact its phosphorylation by LATS1/2. In addition, we tested whether the phosphorylation of YAP by MEKK2/3 affects the interaction between YAP and TEAD. However, MEKK2 and MEKK3 did not interfere the interaction between YAP and TEAD in LATS1/2 DKO HEK293A cells . Together, our results support the functional importance of MEKK2/3 in inhibition of YAP activity through LATS1/2-dependent and -independent manners.Next, we sought to determine the function of YAP phosphorylation by MEKK2/3. Expression of MEKK2 or MEKK3 significantly inhibited both YAP and YAP5SA-mediated TEAD transcriptional activity in WT HEK293A and A and B). Interestingly, signals such as serum starvation and actin depolymerization, which stimulate Hippo activity, also activated MEKK2/3 kinases as shown by the increased MEKK2/3 phosphorylation (A and B). Consistently, LPA, which inhibits Hippo activity, suppressed MEKK2/3 activity (C). Next, we tested whether MEKK2/3 are involved in Hippo signaling. As expected, TNF-induced LATS phosphorylation was significantly compromised in Mekk2/3 DKO NIH3T3 cells (D). Intriguingly, cell contact, serum starvation, and actin depolymerization-induced LATS and YAP-S127 phosphorylation was also greatly reduced in Mekk2/3 DKO NIH3T3 cells and MEKK2/3 DKO HEK293A cells (E–G and A and B). The deletion of Mekk2/3 in NIH3T3 cells resulted in a more profound inhibition on the phosphorylation of LATS and YAP stimulated by various signals. We found that NIH3T3 cells have a higher protein level of MEKK2 and MEKK3, while HEK293A cells express a higher level of MST1/2 and MAP4K4 (C). Previously, we have shown that FGF2 induces the phosphorylation of MEKK3 in NIH3T3 cells (Mekk2/3 completely abrogated FGF2-induced LATS and YAP phosphorylation (D). These results suggest that MEKK2/3 mediate multiple signals to activate LATS1/2.MEKK3 is an essential kinase to mediate NF-κB signal transduction. The deletion of MEKK3 disrupts TNF-induced NF-κB activation . Many strylation , A and Bactivity C. Next, T3 cells D. Intrig E–G and , A and Bd MAP4K4 C. PrevioT3 cells . Thereforylation D. These Mekk2/3 DKO cells even in the presence of TNF (H). Together, these results suggest that MEKK2/3 are required for various signals-induced LATS activation and are involved in Hippo-YAP regulation.YAP phosphorylation at Ser127 by LATS1/2 is an important step for regulation of nuclear/cytoplasmic localization, which is one of the most important mechanisms in physiological regulation of YAP activity , 45. Pree of TNF H. TogethA). Unlike MEKK3, MEKK2 did not interact with CMM2 and CCM3 . Recent studies showed that phosphorylated MST1/2 was recruited to STRIPAK complex through SLMAP for inactivation by PP2A (phosphatase 2A) (A).Loss-of-function mutations in CCM family contribute to CCM disease , 34. CCMFig. 6tase 2A) , 48. Howtase 2A) A.Figure B). Notably, serum starvation or actin depolymerization disrupted the interaction between CCM3 with MEKK3 and CCM2 complex (C). Furthermore, we tested whether MEKK3 formed a complex with STRIPAK at endogenous level. MEKK3 proteins were immunoprecipitated from HEK293A cell lysates. Consistently, STRN3 was detected in the immunoprecipitated complex by anti-MEKK3 antibody, and this association was disrupted by serum starvation and actin depolymerization (D). Notably, endogenous interaction between CCM2 and MEKK3 was also disrupted during the treatments (D). The inconsistency may be caused by the strong interaction between MEKK3 and CCM2 in the overexpression system. MST1/2 and MAP4Ks have been shown to be dephosphorylated and inactivated by PP2A in the STRIPAK complex. To test whether STRIPAK could inactive MEKK3, CCM2 and CCM3 were transfected individually or combinedly with MEKK3. Of note, the phosphorylation of MEKK3 and LATS1 was significantly reduced only when CCM2 and CCM3 were both present with MEKK3 (E). In addition, YAP-S127 phosphorylation was abolished in the presence of CCM2/3 and MEKK3 (E), which indicates the failed activation of LATS1 kinase activity by MEKK3. Consistently, the removal of CCM2, CCM3, or STRN3 increased the basal phosphorylation of MEKK3, and serum starvation could not further increase the phosphorylation of MEKK3 (F and G). Thus, these results indicate that STRIPAK plays an active role in the repression of MEKK3 activity to regulate Hippo pathway through CCM2 and CCM3.Since MEKK3 interacts with CCM2, and CCM2 binds to CCM3, we sought to test whether MEKK3 can form a complex with CCM3 through CCM2 and whether this complex formation is regulated. To test this, MEKK3-HA protein was coexpressed with CCM2-Flag and CCM3-Flag proteins in HEK293A cells. In the immunoprecipitated complex by anti-HA antibody, CCM2-Flag and CCM3-Flag proteins were readily detected by anti-Flag antibody B. Notabl complex C. Furtherization D. Notableatments D. The inth MEKK3 E. In addnd MEKK3 E, which of MEKK3 , F and GH).Taken together, our study uncovers that multiple signals, such as TNF and serum starvation or actin depolymerization-induced STRIPAK regulation, converge on MEKK2/3 to modulate LATS1/2 and YAP/TAZ activity H.via CCM2 and CCM3. Signals such as serum starvation and actin depolymerization disrupt association between MEKK3 and STRIPAK complex and then activate MEKK3 activity. We propose that STRIPAK plays an active role in the regulation of MEKK3 by relaying upstream signals. Taken together, our work has uncovered a previously unrecognized regulation of Hippo pathway via MEKK2 and MEKK3 and provides new insights into molecular mechanisms for the interplay between Hippo-YAP and NF-κB signaling and the pathogenesis of CCM disease.The Hippo signaling pathway plays a central role in regulating cell proliferation, cell fate, and tissue size. LATS1/2 are the primary direct kinases phosphorylating YAP/TAZ in the Hippo pathway. Many studies have revealed that the Hippo pathway can integrate various upstream extracellular and intracellular signals to regulate LATS1/2 activation. Our study adds a new dimension to Hippo regulation. Here, we showed that MEKK2/3 interact with LATS1/2 and mediate TNF-induced LATS activation and YAP/TAZ inactivation. Furthermore, MEKK3 associates with STRIPAK complex in vitro kinase assay in a recent study  positively and Yorkie (Drosophila homology of YAP/TAZ) negatively regulate the innate defense by regulating the transcription of Cactus (Drosophila IκB factor)  . Similaractivity . We haveactivity . In addiactivity . Here, oIt has been known that MEKK3 and CCM signaling play critical roles in regulation of the development and vasculature. CCM formation can be genetically rescued by loss of MEKK3 . How MEK2. Lipofectamine 3000 (Invitrogen) or polythylenimine (PEI) (Polysciences) transfection reagents were used for plasmid transfection. TNF and FGF2 were purchased from Peprotech. HEK293A (3 × 105/well), and NIH3T3 (1 × 105/well) cells were sparsely seeded on six-well plates and treated with 5 ng/ml TNF or 50 ng/ml FGF2 for different times. MG132 (C2211) was purchased from TargetMol. Lysophosphatidic Acid (LPA) (sc-201053) and Latrunculin B (sc-203318) were purchased from Santa Cruz. Cells were treated with Latrunculin B (200 ng/ml) to disrupt the actin cytoskeleton. We generated a kinase-dead mutant MEKK3-KD, which contains S526/T530 to Ala point mutations, and a kinase-dead mutant MEKK2-KD, which contains S521/T523 to Ala point mutations. The rabbit polyclonal antibody against the phosphorylated of YAP-S371 was produced using the synthetic phosphorylated peptides PGM-S(p)-QELRTMT-C as antigen and purified on a phosphopeptide column.HEK293A, HEK293T, NIH3T3, and A549 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. All the cells were cultured at 37 °C in cell culture incubator with humidified environment in 5% COGuide RNA sequences targeting RIPK1, TAK1, MEKK2, and MEKK3 were cloned into plasmid PX459 (Addgene #62988). Constructs were transfected into HEK293A or NIH3T3 cells by PEI transfection reagent. Twenty-four hours after transfection, cells were selected by puromycin (1.5 μg/ml) for 72 h. Single colonies were picked and characterized by immunoblotting. Guide RNA, shRNA, and siRNA sequences are listed in GAPDH. The sequences of the primers are listed in Total RNA from cultured cells was isolated using TRIzol Reagent (Invitrogen) according to the manufacture's protocol. Two microgram total RNA was reversely transcribed using SuperScript III Reverse Transcript Kit (Invitrogen). Quantitative PCR was then performed using SYBR Green 2X PCR Master Mix (Yeasen Biotech) on an Applied Biosystems 7900 system (Applied Biosystems). Target gene threshold cycles  were normalized to HEK293T cells were transfected with pGL3-basic, Gal4/TEAD4-luciferase reporter plasmid together with pRL-TK vector (Promega) as reference control using PEI (Invitrogen). Cells were subjected to luciferase activity measurement as described in dual luciferase reporter assay kit (Promega).Equal amount of proteins was loaded into each well and separated by SDS-PAGE. After blocking in 5% milk or 3% BSA, the membrane was incubated with primary antibody at 4 °C overnight. The membrane was washed and subjected to incubation with the HRP-conjugated secondary antibody for 1 h at room temperature. Western blotting images were captured by ChemiScope5300 (Clinx) with ECL substrate. Antibody information is described in After transfection with indicated plasmids for 24–36 h, cells were lyzed in a lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton NP-40, and 1 mM EDTA with protease and phosphatase inhibitors on ice. Twenty microliter anti-Flag M2 magnetic beads or anti-HA magnetic beads were added to the lysates and incubated at 4 °C overnight under gentle agitation. The beads were washed with washing buffer for five times. Finally, the beads were eluted with 2× SDS loading buffer. The eluted proteins were analyzed by SDS-PAGE and followed by immunoblot analysis.g for 3 min at 4 °C. The supernatant was cytoplasmic fraction. Nuclear pellets were washed in Buffer A, 5 min for three times, and resuspended in 100 μl Buffer B  at 4 °C for 10 min. The supernatant was nuclear fraction after centrifuging at 10,000g for 5 min.Cells were lysed in 100 μl Buffer A  with protease inhibitors cocktail at 4 °C for 15 min. Lysates were cleared by centrifuge at 10,000HEK293T cells were transfected with plasmids expressing Flag-YAP, MEKK3-Myc or HA-ubiquitin. Cells were pretreated with MG132 at 10 μM for 2 h and then treated with TNF at 5 ng/ml for 1 h. Cell lysates were harvested with RIPA buffer  and incubated with 20 μl anti-Flag M2 magnetic beads at 4 °C overnight. The beads were washed for five times. Bound proteins were boiled in 2× SDS loading buffer and analyzed by SDS-PAGE followed by immunoblot analysis.E. coli by glutathione agarose slurry. Purified proteins were washed with kinase washing buffer  three times and once with kinase assay buffer . Purified kinase and YAP proteins were mixed with ATP-γ-S (500 μM) or ATP (500 μM) in kinase assay buffer. After 1 h kinase reaction at 30 °C, EDTA  was added to terminate the reaction at 30 °C for 5 min. PNBM  (2.5 mM) was added at 25 °C for 40 min to form a thiophosphate ester side chain in the reaction with ATP-γ-S. Western blot was performed using anti-YAP-S371 phospho-specific antibody or anti-thiophosphate ester antibody to analyze the kinase activity.HEK293T cells were transfected with Flag-tagged MEKK3 plasmids and then lysed in RIPA buffer. Flag-tagged MEKK3 proteins were purified by anti-Flag antibody-conjugated magnetic beads. GST-tagged YAP proteins were purified from HEK293A or NIH3T3 cells were plated on coverslips. Cells were transfected with plasmids by PEI or treated with TNF at 5 ng/ml for 0, 30, and 60 min and were then fixed with 4% PFA on ice for 20 min. After fixation, cells were then washed with PBST , followed by blocking with 3% BSA at room temperature for 30 min. Cells were incubated with primary antibodies diluted in 3% BSA overnight at 4 °C. After three washes with PBST, cells were incubated with secondary antibodies in 3% BSA for 1 h. Nuclei were stained with 0.5 μg/ml of DAPI at room temperature for 10 min. Cells were mounted with ProLong Gold Antifade  and viewed under a Nikon NI-U fluorescent microscope.t test and one way ANOVA with Dunnett's test were used for pairwise comparisons and multigroup comparison respectively. p values <0.05 were considered to be significant. All analyses were performed with GraphPad Prism software (Version 6.0). The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. Western blot shown is representative of at least two independent experiments.Student's All the data are included in the article.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Here, we report the anti-PEDV effect of the broad-spectrum inhibitor GC376 (targeting 3Cpro or 3CLpro of viruses in the picornavirus-like supercluster). GC376 was highly effective against the PEDV 3CLpro and exerted similar inhibitory effects on two PEDV strains. Furthermore, the structure of the PEDV 3CLpro in complex with GC376 was determined at 1.65 Å. We elucidated structural details and analyzed the differences between GC376 binding with the PEDV 3CLpro and GC376 binding with the transmissible gastroenteritis virus (TGEV) 3CLpro. Finally, we explored the substrate specificity of PEDV 3CLpro at the P2 site and analyzed the effects of Leu group modification in GC376 on inhibiting PEDV infection. This study helps us to understand better the PEDV 3CLpro substrate specificity, providing information on the optimization of GC376 for development as an antiviral therapeutic against coronaviruses.Porcine epidemic diarrhea virus (PEDV), being highly virulent and contagious in piglets, has caused significant damage to the pork industries of many countries worldwide. There are no commercial drugs targeting coronaviruses (CoVs), and few studies on anti-PEDV inhibitors. The coronavirus 3C-like protease (3CL Alphacoronavirus in the family Coronaviridae and causes severe diarrhea, vomiting, dehydration and high mortality in neonatal piglets, resulting in severe agricultural loss [Porcine epidemic diarrhea virus (PEDV), which was first observed in Europe in 1971 [ral loss ,3. Sinceral loss . Howeverral loss ,5,6. Theral loss . Furtherral loss ,7. Althoral loss . Thus, ipro; main protease, Nsp5) at 11 conserved sites, which are essential for viral replication [pro is an appealing target in the design of anticoronavirus therapies [pro proteins of different coronaviruses share higher sequence identity and have a more conserved catalytic core than the spike proteins of the same coronaviruses [pro an ideal broad-spectrum antiviral target [Like all the other coronaviruses (CoVs), PEDV has a RNA genome that encodes two polyproteins, pp1a and pp1ab ,10,11. Tlication ,13. Thusherapies ,16,17,18aviruses ,20,21,22l target ,24,25,26l target ,28,29,30pro employs conserved cysteine and histidine residues, which serve as the principal nucleophile and general acid–base catalyst, respectively, at its catalytic site [pro recognizes a conserved cleavage site containing a hydrophobic residue (preferably L) at the P2 position, a Q at the P1 position, and a small aliphatic amino acid residue  at the P1′ position [pro structures have made it possible to design specific inhibitors against coronavirus replication, and some of these inhibitors show excellent broad-spectrum anti-coronavirus effects [pro shares a 62% sequence identity with FIPV 3CLpro.The coronavirus 3CLtic site ,10,31. 3position ,33. Rese effects ,31,34,35 effects ,37. Nota effects . In addipro. The antiviral effects of the inhibitor were studied using two PEDV strains, namely, CV777 and YN144. Finally, we determined the structure of the PEDV 3CLpro in complex with GC376 and compared this structure with other available 3CLpro structures. Our study provides a structural model for the development of anti-PEDV drugs and further enriches structural knowledge for the optimization of broad-spectrum anticoronavirus drugs.In this study, we detected the inhibitory effects of GC376 on the PEDV 3CLwww.labnetwork.com, Shanghai 200131, China) with purity higher than 95%. In Target 1, Leu of GC376 is replaced with Met, which has a longer side chain; in Target 2, Leu of GC376 is replaced with Phe, which has a side chain with a larger volume. The compound was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 100 mM and stored at −80 °C.GC376 A and itsAccession No. KT021232, [African green monkey cells  were cultured with Dulbecco’s minimum essential medium  supplemented with 10% fetal bovine serum . The viruses used in this study were the attenuated strain YN144  and theT021232, . Notablypro was described in a previous study [6-tag was inserted into the pET42b (+) vector. Proteins were induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 27 °C for 7 h. A His TrapTM HP column  and a 120-mL Superdex 200  column were used for protein purification.The strategy for plasmid construction, protein expression and purification of the PEDV 3CLus study . Brieflypro, was used in FRET assays. All reactions were performed in a solution of 20 mM HEPES, 50 mM NaCl, 0.4 mM EDTA, 30% glycerol and 4 mM DTT at pH 8.0 in a total volume of 100 μL. The enzyme concentration used in FRET assays was 100 nM, and the substrate concentration was 40 μM. The concentrations of GC376 ranged from 0.03 μM to 80 μM. The fluorescence units upon cleavage were monitored every 5 min for 30 min at 485 nm, with excitation at 340 nm. Relative fluorescence units (RFU) were determined by subtracting the background value (substrate control well without the protease) from the fluorescence values. Finally, the 50% inhibitory concentration (IC50) was calculated using GraphPad Prism5  to assess the inhibition ratios at different inhibitor concentrations.The fluorescent peptide substrate “Dabcyl-YNSTLQ↓AGLRKM-E-Edans” , which contains the N-terminal cleavage site of the PEDV 3CL50) measurement, the 50% cell death toxic concentrations (CC50) of GC376 in Vero cells were determined. Briefly, confluent cells grown in 96-well plates were treated with various concentrations (50–200 μM) of GC376 for 72 h. All the wells, including control wells, contained 0.2% DMSO. Cell cytotoxicity was measured using the Celltiter-Glo Luminescent Cell Viability Assay reagent . For EC50 measurement, two PEDV strains (CV777 and YN144) were used. When cells were grown to 80%–90% confluence, the virus was added at a multiplicity of infection (MOI) of 0.01 with a series of GC376 concentrations . All the wells, including the virus and cell control wells, contained 1% DMSO. Cell viability was measured as above at 12 h and 36 h for YN144 and CV777, respectively. Finally, the EC50 was calculated using GraphPad Prism5  to assess the inhibition ratios at different inhibitor concentrations.Before the 50% maximal effect  on ice. The supernatants were used for Western blot assays. After SDS-PAGE, the proteins were transferred to PVDF membranes . After blocking with 5% skimmed milk at room temperature for 3 h, the membranes were incubated with an anti-PEDV nucleocapsid (N) protein monoclonal antibody and an anti-GAPDH monoclonal antibody  for 2 h. Then, the membranes were incubated with an HRP-conjugated secondary antibody  for 40 min. The membranes were visualized using an enhanced chemiluminescence system , and the nucleocapsid protein level was normalized to the GAPDH protein level.Vero cells were seeded in 6-well plates at a density of 1 × 104 cells per well. YN144 was inoculated into the wells at an MOI of 0.01, and GC376 was added at a series of concentrations at the same time. Indirect immunofluorescence assays were performed when 60% of the cells died. Briefly, cells were washed with PBS before fixation by 4% formaldehyde in PBS, treated with 0.1% Triton X-100, and blocked with 2% BSA. After blocking, the cells were incubated with the primary antibody and a FITC-labeled secondary antibody  in the dark. Finally, the cells were examined under fluorescence microscopy after staining with DAPI.Vero cells were seeded in 96-well plates at a density of 1 × 10pro for GC 376 soaking was obtained as described in a previous study [v/v) as a cryoprotectant and then flash-frozen in liquid nitrogen. All data collection was performed at beamline BL17U at the Shanghai Synchrotron Radiation Facility (SSRF) using a MAR 225 CCD detector (MAR Research). The data sets were indexed, integrated and scaled using HKL-3000  [pro (PDB identifier 4XFQ) as a starting model. Manual model building was performed using Coot  [The crystal of the PEDV 3CLus study . GC376 wVA, USA) . The strVA, USA)  using thxon, UK) , and thexon, UK) . Refinempro from the FJZZ strain, the sequences for 3CLpro from the YN144 strain and CV777 strain were identified to differ by one and six residues, respectively. All of the different residues were far from the substrate-binding site of the PEDV 3CLpro  and might not cause obvious differences in enzymatic activities; thus, we used the PEDV FJZZ 3CLpro stored in our laboratory for inhibition assays. The results indicated that GC376 exerted strong inhibitory effects on the PEDV 3CLpro with an IC50 of approximately 1.11 μM  much faster than CV777 (12 h and 36 h respectively); however, GC376 showed similar inhibitory effects with different treatment times for the two viruses, which indicates that GC376 quickly enters the cell and blocks viral 3CLpro.Before determining the inhibitory effects of GC376 on viruses in Vero cells, we tested the cytotoxicity of the compound. GC376 had no cytotoxicity up to a concentration of 200 μM (data not shown). GC376 at a series of concentrations was used to evaluate the suppression of two PEDV strains. The ECectively . The res50 of approximately 12.5 μM, which was consistent with that of the cell-based IC50 measurement assays. Since GC376 showed similar effects against the two PEDV strains, we performed IFA with one of the PEDV strains, YN144. Compared with the non-drug-treated cells, GC376 reduced virus infection at 1.56 μM . Investigation into the substrate-binding site revealed a prominent electron density for GC376 in two monomers, especially in subunit A . As the 6-membered aromatic ring of GC376 occupied the place of the PEDV 3CLpro substrate P3 residue, whose side chain usually points to the solvent, it contributed little to the interface  yielded root mean square deviations (RMSDs) of 0.31 Å and 0.18 Å between the Cα atoms for chains A and B, respectively. This finding indicated that no large conformational change in the overall structure occurred upon ligand binding. However, examination of the substrate-binding pocket between chains A and B of the PEDV 3CLpro-GC376 complex revealed a structural difference in the loop composed of residues 44–52 (ASSTTSTID). Interestingly, there was only electron density (contoured at 1.0 σ) for the 46–48 residues (STT) of chain A but not for chain B. This result suggests that the part of the loop is flexible and that its orientation directly influences the entrance size of the substrate-binding pocket. Thr 47 in the loop and Asn 141, which is located on another side of the binding pocket, both protrude into the pocket, forming a narrow entrance with the width of ~8 Å . To determine the preferred P2 residues of PEDV 3CLpro, fluorescent peptide substrates with different P2 residues were synthesized. Except for Val and Met, several other residues with different side chain sizes were introduced into the substrates (pro.Superimposition of the PEDV nsp5-GC376 and PEDV nsp5-peptide complex (PDB: 4ZUH)  indicatebstrates B. FRET a50 of the two compounds fir YN144 infection was measured to evaluate optimization effects. Targets 1 and 2 yielded EC50 values of 65.7 μM and 80.0 μM respectively  was stable, and water-mediated contacts were observed between Thr 47 and GC376 in the TGEV 3CLpro–GC376 complex, which have also been found in the norovirus (NV) 3CLpro and picornavirus (PV) 3CLpro in complex with GC376 [pro-GC376 complex. b. The Asn 141 residue, which has a side chain that protrudes into the pocket, resulted in a narrower entrance in the PEDV 3CLpro than that in the TGEV 3CLpro, which has an Ala 141 residue instead , and the structure of TGEV 3CLtermined . We compor GC376 A. The RMor GC376 B. Some oth GC376 ; however instead . The twoViral proteases play a vital role in the viral lifecycle and are confirmed to be ideal antiviral targets. Several remarkably potent drugs targeting individual viral proteases have been developed against important human pathogens, such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV) ,46,47,48pro responsible for viral polyprotein cleavage. Porcine epidemic diarrhea virus (PEDV), an α coronavirus, is one of the major threats to the swine industry worldwide. The development of antiviral drugs targeting 3CLpro is an important strategy to prevent PEDV epidemics. The global outbreak of SARS in 2003 accelerated study on anti-coronavirus inhibitor development, and many researchers have been committed to developing broad-spectrum antiviral inhibitors against coronavirus 3CLpro.This strategy is also applicable to coronaviruses, which encode a 3CL50 of 1.11 ± 0.13 μM for GC376 against the PEDV 3CLpro, which was slightly higher than that against the transmissible gastroenteritis virus (TGEV) 3CLpro (0.82 ± 0.47 μM) and lower than that against the SARS-CoV 3CLpro (4.35 ± 0.47 μM) [In our study, a series of available coronavirus inhibitors were applied in FRET-based assays to identify an effective inhibitor of PEDV. GC376, a dipeptidyl bisulfite adduct salt, worked best and was selected as the subject of subsequent experiments. Biochemical assays yielded an ICpros from the CV777 and YN144 strains revealed seven different residues. However, both EC50 and Western blot assays showed similar antiviral effects for GC376 on the two virus strains . However, most of the residues involved in GC376 binding are conserved. Only the loop (46-49) and entrance size show some differences  in piglets. In general, GC376 exerted an excellent inhibitory effect on different PEDV strains by targeting its 3CL