Wedemonstrate that the p-amino-calix[4]arene–GPGKLVFFconjugate alters the Aβ42 aggregation pathways bypreventing Aβ42’s conformational transitionfrom random coil to β-sheet with concomitant changes of theaggregation kinetic profile as evidenced by circular dichroism (CD),thioflavin T (ThT), and dynamic light scattering (DLS) measurements,respectively. High resolution mass spectrometry (HR-MS) confirmeda direct interaction of the p-amino-calix[4]arene–GPGKLVFFconjugate with Aβ42 monomer which provided insightinto a possible working mechanism, whereas the alteration of the Aβ42’s fibrillary architecture, by the calix-peptide conjugate,was further validated by atomic force microscopy (AFM) imaging. Finally,the herein proposed compound was shown to be effective against Aβ42 oligomers’ toxicity in differentiated neuroblastomacells, SH-SY5Y.Alzheimer’sdisease (AD) is a progressive neurodegenerativecondition affecting people in the elderly. Targeting aggregation ofβ-amyloid peptides (Aβ) is considered a promising approachfor the therapeutic treatment of the disease. Peptide based inhibitorsof β-amyloid fibrillation are emerging as safe drug candidatesas well as interesting compounds for early diagnosis of AD. Peptideconjugation via covalent bond with functional moieties enables theresultant hybrid system to acquire desired functions. Here we reportthe synthesis, the structural characterization, and the Aβ AD represents one of the “protein misfoldingdiseases” with the highest socioeconomic impact30 Conjugation can also be used to deliver a cytotoxic payload or imagingagent to specific cell types targeted by the peptide.31 Calix[n]arenes are macrocyclic polyphenolsproposed as molecular scaffolds for different fields of application.32 These macrocycles due to the peculiar structureand synthetic versatility have gained great interest in supramolecularchemistry. The calix[4–8]arene oligomers possess a hydrophobiccavity able to host a variety of guests and can cluster and spatiallyorganize multiple ligands, providing rigid or flexible constructssuited for molecular recognition events. The low toxicity and immunogenicityexhibited by a variety of calix[n]arene derivativeshave advanced these macrocycles to applications in biomedical andpharmaceutical fields.33 Calix[n]arene derivatives have been proposed as anticancer,34 antibacterial,35 syntheticvaccines,36 imaging agents,37 and drug38 and gene39 delivery systems. Opportunely functionalized,the calix[n]arenes have also been exploited as biomimeticmodels to better understand relevant biological processes underlyingthe protein–protein or protein–carbohydrate interactions.40 Yet, calix[n]arenes have beensuccessfully used as novel compounds for protein detection40 and protein modulation.41 However, despite the wide use of calix[n]arenesin several fields of application, very few papers have dealt withthe interactions of calix[n]arene derivatives withAβ. Wang et al. described p-sulfonate-calixareneswith anti- and disaggregating effect on Aβ42 throughnonspecific hydrophobic interactions.42 Guo et al. synthesized a nanoassembly consisting of multiple unitsof a p-sulfonate calix[4]arene and a β-cyclodextrin,which by means of the multiple complexing cavities exposed on thenanoassembly surface bind Aβ42, inhibiting its aggregationor inducing fibril disaggregation.43 Wedetermined that endowing the calixarene macrocycle with an Aβrecognition motif would generate a more refined tool to contrast thedetrimental effects of the aggregated forms of Aβ peptides.Thus, we resorted to the design and synthesis of a new calix[4]arene–peptideconjugate composed of a p-amino-calix[4]arene derivativebearing a GPGKLVFF peptide sequence at the macrocycle lower rim. Wedescribe herein the synthesis and structural characterization of thishybrid system, alongside its capability to hamper Aβ42 aggregation in vitro. The study has been carriedout using a variety of spectroscopic techniques including circulardichroism (CD), thioflavin T (ThT) fluorescence, dynamic light scattering(DLS), and atomic force microscopy (AFM) imaging. High resolutionmass spectrometry (HR-MS) was employed to point out direct interactionof the p-amino-calix[4]arene-GPGKLVFF conjugate withthe Aβ42 monomer. We also aim at providing in vitro experimental evidence of the ability of this novelconstruct to prevent the Aβ oligomer cytotoxic effects on differentiatedSH-SY5Y neuronal cultures. We demonstrate that the calix–peptideconjugate is per se nontoxic to neuronal cells andthis allows its potential use as therapeutic agent in AD.Therefore,conjugation has emerged as a popular mechanism to modifyor enhance the properties of a peptide drug candidate.n]arene inhibitorsof Aβ amyloid fibrillogenesis, we thoughtthat joining an Aβ recognition peptide moiety to the frameworkof a water-soluble p-amino-calix[4]arene derivativewould result in a new hybrid construct able to efficiently interactwith Aβ peptides and therefore hampering peptide chain’sself-assembly into oligomeric/fibrillary toxic species. We chose theGPGKLVFF sequence because of the established ability of the KLVFFmotif to recognize the homologous region of the parent full-lengthAβ peptide. The additional GPG tripeptide was inserted to reduceany eventual propensity to self-aggregation of the calix–peptideconjugate. The p-amino-calix[4]arene scaffold shouldconfer enhanced water solubility to the resultant calix–peptideconjugate, at the same time establishing multiple noncovalent contactswith the aromatic and anionic amino acid residues of the Aβpeptide. Moreover, it is expected that the calix–peptide conjugatewould generate a steric hindrance between adjacent peptide chainsthereby disturbing the Aβ’s self-assembly process. Comparedto other peptide-conjugated systems, the peptide–calixarenedifunctional compound can offer some advantages deriving from theknown complexing properties of the calixarene cavity, in additionto the antiamyloidogenic action of β-sheet breaker pentapeptideKLVFF as a “binding element” for targeting Aβ.The calixarene moiety might also synergically assist the peptide ligandin both Aβ monomer stabilization and protection of neurons fromAβ oligomeric insult. The calix–peptide hybrid systemmight in principle be useful for delivery purposes of AD active compoundsor imaging agents for theragnostic application.In search of functional calixarene scaffold was accomplished in solution and inthe presence of PyBop as a condensing agent. The selective removalof the Dde and Boc protecting groups, by sequential treatment withhydrazine and trifluoroacetic acid respectively, gave the compound 5 whose molecular identity was confirmed by combined MALDI-TOF-MS,HR-ESI-MS and 1D/2D NMR spectra .The procedurefor the preparation of the calix-peptide conjugate , werecarried out to examine the effect of these derivatives on the Aβ42’s β-sheet conformational transition. 42 alone and in the presence of compound 5. It is clear from 5, the Aβ42 sample (5 μM) undergoes to an almost complete β-sheetconformational transition after 72 h. In fact, the freshly preparedAβ42 sample displays clear negative dichroism below200 nm that is typical of a randomly coiled peptide chain. This curveprofile gradually changes toward the β-sheet pattern, as theincubation time proceeds. The CD spectrum recorded at 72 h incubationtime shows positive ellipticity at 190 nm along with a negative signalat 218 nm, thereby indicating the presence of β-sheet peptidestructures.48 This pattern does not changesignificantly after this time, indicating that Aβ42 almost reaches the final state after 5 days of incubation. The CDspectra recorded in the presence of 5, either at equimolaror 5 mol excess, never display the characteristic β-sheet pattern.More interestingly, the observed CD spectra recorded at 5-fold molarexcess always exhibit strong negative ellipticity at 190 nm alongwith no apparent inflection of the negative band around 218 nm inthe considered interval of time.48 Theexperimental CD curves were subjected to deconvolution analysis usingthe CONTINN and CDSSTR algorithms.49 In t = 0 or t = 120 h is reported. The valuesof the determined random coil secondary structure are reported in Table S1. It is apparent that at t = 120 h a higher percentage of random coil conformation is maintainedin the presence of the derivative 5 with respect to Aβ42 control. The CD spectra of the calix–peptide conjugate 5 alone were recorded in the same experimental conditions.These spectra are characterized by a generalized low CD amplitudethat does not change with time . All the above indicates that 5 significantly inhibitsthe Aβ42’s β-sheet conformational transitiontypically associated with the fibrillogenesis process.47 In other words, the CD results suggest that 5 well preserves the Aβ randomly coiled monomer fromits recruitment into potentially toxic aggregates. We wanted alsoto evaluate the ability of the calix[4]arene macrocycle to interferewith the random coil/β-sheet conformational transition of Aβ42. We then acquired a new set of CD measurements, under thesame experimental conditions as above, on a sample of Aβ42 and the p-amino-calix[4]arene derivative 1 in both 1:1 and 1:5 molar ratios. CD data showed that compound 1 is able to interact with Aβ42 , although to a lesser extent than 5. This also became evident from the deconvolution analysis. However,the comparison with the effects generated by the synthesized conjugate 5 allows us to point out some differences. Unlike the caseof compound 5, at the 1:1 ratio with Aβ, the unconjugatedGPGKLVFF peptide is able to accelerate the fibrillogenic process.This turns evident in the CD experiments . A typical CD profile of the β-sheet conformationwas observed after 48 h coincubation . The acceleration of the aggregation ratecaused massive precipitation, and we were not able to acquire CD spectraat 72 h . The CD data obtainedat a 1:5 ratio indicated that the free GPGKLVFF can slow down theaggregation process of Aβ but, in any case, to a lesser extentthan the one observed in the presence of the calix–peptideconjugate at both 1:1 and 1:5 ratios. In conclusion, the CD studysuggests that compounds 1 and free GPGKLVFF can affectthe Aβ42 propensity to adopt β-sheet conformation.Importantly, the CD experiments clearly indicated the superiorityof the hybrid system 5 in inhibiting Aβ42 aggregation compared to the separate calix or peptide moieties.The conformational transition froma random coil to a β-sheet structure is the crucial step forthe fibrillogenesis of Aβith time S11. All 42 aggregation was also monitored by ThT fluorescence.ThT is a fluorophore that enhances its fluorescence intensity uponbinding to amyloid fibrils.50 Fluorescenceintensity was monitored at 482 nm for 67 h and at 37 °C. Theresults are presented in 42 displayed a rapid increase of the ThT fluorescence, as expectedfrom the inherent propensity to amyloid formation of this peptide.It is also evident that the curve profile observed displays just ahint of initial nucleation phase (t = 0) might be the responsible of suchbehavior. It is assumed that the lower fluorescence intensity we observedin the presence of the inhibitors at either equimolar ratio or 5-foldmolar excess is related to a lesser amount of Aβ42 fibrillary aggregated form. The antifibrillogenic activity of theconjugate peptide 5 is evident at equimolar ratio . An alternative interpretation impliesthe ability of free GPGKLVFF compound to become an integral part ofThT positive heterofibrils with Aβ51 .Such an effect becomes even more apparent at 5-fold molar excess.As one can see, a very long lag phase, along with a negligible increasingof ThT fluorescence intensity, testifies an almost complete inhibitionof fibril formation . The end stage of Aβ aggregation was analyzed after 120 h,and the formation of larger aggregates was observed. In particular,the DLS profile indicated the presence of particles with increaseddiameter values (>100 nm) as the most abundant scattering entities,along with a very low percentage of bigger aggregates with size inthe micrometric range. The data collected in the presence of 5 clearly indicated the reduction of the dimension of Aβ42 aggregates both at the initial stage and end point of monitoring.In fact, at t = 0, the majority of the scatteringparticles in solution displayed a diameter size of around 45 nm  formed small aggregateswith a mean hydrodynamic diameter around 8 nm at t = 0. Larger aggregates (diameter > 300 nm) were seen at t = 120 h . At 20μM, compound 5 forms nanoaggregates with a meanhydrodynamic diameter of 22 nm and ζ potential 50 ± 2 mV.We resorted to DLS measurementsto determine the average size of the aggregates in solution and tomonitor their growth over time, either in the absence or in the presenceof nd 45 nm 4, wherea42 aggregates. 42 (5 μM) alone formeda fibrillary network with an average fibril height of 2.65 ±0.44 nm. The copresence of 5, at the same concentration(5 μM), during the Aβ42 incubation time of120 h, clearly inhibited the development of amyloid fibrils, and onlysmall amorphous aggregates are detected with sizes of 4.30 ±1.33 nm  studiesrevealed the morphologies of Aβ1.33 nm 5b.Stati5 were added to the Aβ42 sample solution. The Aβ42 sample and the1:5 mixtures of Aβ42/5 and Aβ42/GPGKLVFF, were monomerized using the protocol reported inthe 2O, to obtain a Aβ42 finalconcentration of 5.5 × 10–6 M (1% HFIP). TheESI-MS spectrum of Aβ42 reported in m/z signals at 1129.33 and 1505.43 corresponding to [Aβ42]4+ and [Aβ42]3+,respectively. A closer inspection of the ESI-MS spectrum revealeda signal at m/z = 1806.52 correspondingto the mass of the dimeric peptide in the +5 charge state , whereas m/z signals 1507.79 and 2010.06 correspondingto the +4 and +3 charge state of Aβ42/5 adduct were observed. Similar effects were seen in the mass spectrumof the Aβ42/GPGKLVFF sample . Here, m/z signals1345.20 and 1793.27 can be assigned to the [Aβ42/GPGKLVFF]4+ and [Aβ42/GPGKLVFF]3+ adducts,respectively, with concomitant loss of the dimeric form of Aβ42.ESI-MSmeasurements werecarried out to verify the formation of molecular adducts when GPGKLVFFand the relative conjugate ge state 6, inset.42 and GPGKLVFFor 5. Nevertheless, this study reveals that the dimericform of Aβ42 is no longer detectable in the MS spectrumwhen either GPGKLVFF or 5 is present in the sample solution.Such evidence suggests, once more, that these interactions occur inthe solution phase and may play a role in the early events of theAβ42 aggregation process. As reported in our previousworks,27 the identification of proteolysis resistantpeptides fragments by mass spectrometry may reveal the binding regionof Aβ42 to specific molecules. Indeed, cleavage ofthe peptide bonds by a protease is rapid in easily accessible unstructuredregions of a polypeptide, whereas the steric hindrance of other moleculesat the cleavage sites should affect the rate of hydrolysis. We analyzedthe Aβ42 peptide fragments generated after 2 h oftrypsin digestion. This enzyme selectively catalyzes the hydrolysisof peptide bonds at the C-terminal side of lysyl and arginyl residues.As a matter of fact, the peptide fragments Aβ(1–5), Aβ(6–16),and Aβ(17–28) were detected in the ESI-MS spectrum , indicating the cleavages expectedoccur at positions 5, 16, and 28 of Aβ42. The peptidefragment Aβ(1–16) was also observed. It could be relatedto the conformational features of Aβ42 that affectthe accessibility to the Arg-5-His-6 cleavage site (Scheme S1). The absence of signals corresponding to Aβ42 suggested that protein digestion by trypsin was totallyaccomplished after 2 h  recorded after 2 h of trypsin digestion in the copresence of 5. Similar results were observed in mass spectra of Aβ42/GPGKLVFF mixture (data not shown). It is important to notethat both GPGKLVFF and 5 were also subjected to trypsindegradation indicating that these peptides do not inhibit enzyme’sactivity. Therefore, it can be envisioned that the interaction ofGPGKLVFF or 5 with Aβ42 affects theprocessing of the amyloid protein by trypsin. Oxidized forms of Aβand related fragments were seen in the MS spectra. Oxidation of peptidesand proteins during electrospray ionization may occur because thegeneration of ions in ESI source is related to an electrochemicalprocess.53Despite the mass spectrometricresults were deduced from the gas-phasesystem, they nicely support what was observed in solution in termsof direct interactions between Aβtrum see S19, indind Aβ17–2 were det5 for its biologicalactivity in order to (i) exclude any potential toxicity to neuronalcells and more interestingly (ii) assess its ability to prevent oligomerstoxicity. For this purpose, we used the neuroblastoma cell line, SH-SY5Y,fully differentiated according to a well-established protocol (seethe trans-retinoic acid (RA) andthe consequent acquisition of a neuronal-like phenotype, we exposedcells to increasing concentrations of compound 5  for 24 h  and cellular response, after24 h exposure. Hence, we tested the ability of compound 5 to protect neurons from the Aβ42 oligomer cytotoxicinsult. Aβ oligomers (oAβs) were obtained from freshlyprepared solution of Aβ42, incubated for 48 h at4 °C in the presence or in the absence of the calixarene–peptideconjugate 5.Based on theprevious promisingresults, we tested the peptide conjugate  seethe . After pfor 24 h 9. The pe5. We have alreadyreported on the antioligomerization activity of a trehalose conjugatedLPFFD peptide.54 The newly synthesizedcompound shows a similar interesting effect of the Aβ derivedpentapeptide with a better trend, making this functionalization evenmore promising also in the light of calix[4]arenes ability to be potentiallyloaded with selected drugs. The biological effect observed on SH-SY5Ydifferentiated cells has been also confirmed by SDS-polyacrylamidegel experiments. The same solutions used to treat the cells were infact loaded onto 4–12% gel polyacrylamide to assess the amountand size of Aβ42 aggregates formed in the presenceof the compound 5 or its related controls. Two differentconcentrations of Aβ42 have been used and each peptideadded with a molar ratio of 1:5. Results show that, as expected, Aβalone during 48 h incubations at 4 °C was able to give rise tothe typical migration pattern in which low molecular weight size  and high molecular weight (HMW: >50kDa oligomers) size are observed . Coincubation with compound 5 strongly decreases theHMW band signal, which confirmed that our compound interferes withAβ aggregation.Aβ oligomers obtainedfollowing the incubation were addedat the final concentration of 2 μM, alone or in combinationwith all of the compounds at the molar ratios of 1:1 and 1:5. Cells,were then exposed for 48 h to each coincubated mixture and cell viabilitywas quantified by MTT assay. As shown in 42 through a synergistic action of both calixareneand peptide moieties that can be considered as β-sheet breakerelements. This is an interesting example of a small construct beingable to preserve the nontoxic monomeric species of Aβ42 from being recruited into oligomeric/fibrillar aggregated forms,as demonstrated by circular dichroism and ThT fluorescence. The lackof toxicity, combined with the significant protective action on neuronalcells, further points toward a considerable therapeutic potentialof this novel construct. Moreover, given the already established pharmaceuticalfunctions of calixarenes in drug delivery, our findings suggest thatthe conjugate has great potential as a vehicle for the targeted deliveryof additional therapeutic agents in AD. In fact, due to the presenceof calix[4]arene and peptide moieties, multiple potential actionscan be expected, and the possibility that the calixarene cavity offersto host and transport a variety of guests, such as drugs or imagingagents, opens up interesting perspectives for further investigationsin diagnosis and therapy for amyloid related diseases. The calix[n]arene family offers a variety of macrocyclic scaffoldsdiffering in size and structural conformation that, together withthe versatility and ease of functionalization, may provide an additionaltool for the development of other peptide–calixarene conjugatesfor Aβ aggregation inhibitors.Inthe present work, we have described the design, synthesis, andbiophysical properties of a new calixarene–peptide constructthat combines the Aβ recognition ability of the GPGKLVFF sequencewith the host and recognition properties of the calixarene macrocycle.This novel compound is able to prevent cross-β-sheet elongationof Aβ1(42 was obtained from Bachem (Switzerland). MALDI-TOF spectra were recordedon the SCIEX TOF/TOF 5800 instrument. The MALDI-MS spectra were carriedout using α-CHCA as a matrix with a thin layer deposition method.Lyophilized samples (0.1 mg) were dissolved in 100 μL of 1:1:0.01acetonitrile/water/TFA. Sinapinic acid (SIN) and α-CHCA wereprepared by dissolving 4–8 mg/vial of matrices in 1 mL of 50%acetonitrile in 0.3% TFA and 1 mL of 30% acetonitrile in 0.3% TFA,respectively. Standard kits were used to calibrate the mass scaleof the MALDI mass spectrometer. The Peptide Mass standard kit includesdes-Arg1-bradikynin, angiotensin I, Glu1-fibrinopeptide B, ACTH (Clip1–17), ACTH (Clip 18–39), and ACTH (Clip 7–38),and it was used to cover a mass range of 800–4000 Da.All the reagents wereof analytical grade. Calix[4]arene derivative 1 and GPGK1H NMR (400.13 MHz), 13C NMR (100.61 MHz),and 2D NMR spectra were acquired on a Bruker Avance 400 spectrometer.Chemical shifts (δ) are expressed in parts per million (ppm),referenced to the residual methanol peak; coupling constant (J) values are given in Hz.2O/TIS (95:2.5:2.5v/v/v). Crude peptides were recovered by precipitation with freshlydistilled diethyl ether. The purification of crude GPGK(Dde)LVFF wascarried out by preparative reversed-phase HPLC using a SHIMADZU LC-20Achromatography system equipped with a SPD-M20A photodiode diode arraydetector with detection at 222 and 254 nm. A Kinetex C18 250 ×21.10 mm  column was used. Thepeptides were eluted at a flow rate of 10 mL/min according to thefollowing protocol: from 0 to 5 min isocratic conditions in 95% solventA (H2O containing 0.1% TFA) followed by a 20 min lineargradient from 5 to 70% B (CH3CN containing 0.1% TFA) andthen 5 min isocratic conditions in 70% B. Fractions containing thedesired product were collected and lyophilized. Sample identity wasconfirmed by ESI-ORPBITRAP MASS. Calculated mass: 1026.59; observed:[M + H]+ = 1027.59; [M + 2H]2+ = 514.30.The peptide was synthesizedusing the microwave assisted solid phase peptide synthesis strategyon a Liberty Peptide Synthesizer (CEM). The peptide chain assemblywas carried out on a NovaSyn TGR resin (substitution 0.22 mmol/g)using the Fmoc chemistry method. All Fmoc-amino acids were introducedaccording to the TBTU/HOBT/DIEA or COMU activation methods. All syntheseswere carried out under a 4-fold excess of amino acid. Removal of Fmocprotection during synthesis was achieved by means of 20% piperidinesolution in DMF. The following instrumental conditions were used foreach coupling cycle: microwave power 25 W, reaction temperature 75°C, coupling time 300 s. The instrumental conditions used forthe deprotection cycles were: microwave power 25 W, reaction temperature75 °C, deprotection time 180 s. The peptides were cleaved offfrom the resin using a mixture of TFA/H1  dissolved in passed through basic alumina CH2Cl2 (8 mL), di-tert-butyl dicarbonate  was added. The mixture was stirred at rt overnight,then a 5% aq NaHCO3 solution (50 mL) and CH2Cl2 (50 mL) were added. The organic phase was washed with5% aq NaHCO3 solution (50 mL) and water (50 mL × 2).After removal of the solvent under vacuum, the solid residue was washedwith hexane and recovered by filtration to give pure compound 2 . 1H NMR  δ: 0.92 , 0.98 , 1.26 , 1.46, 1.49,, 1.56 , 1.85 , 3.08 and4.37 , 3.14 and 4.57 , 3.71 , 3.80 , 4.17 , 4.64 , 6.03 , 6.26 , 6.43, 6.81 .To compound 2 was suspended in THF (10 mL) in the presence of water (2 mL) and KOH. The resulting mixture was refluxed and stirredfor 3 h. The pH was adjusted to 4 with aq 1 N HCl. The product wasextracted with CH2Cl2, and the organic layerwas dried over Na2SO4 and evaporated to givepure compound 3 . 1H NMR  δ: 0.91 , 1.02 , 1.42, 1.51, , 1.88, 3.12 and 4.40 , 3.18and 4.45 , 3.73 , 3.95 , 4.68 , 6.58 , 7.08 .Compound 3 (0.018 mmol) dissolved in 2 mL of dry DMF, pyBop  and DIPEA  were added. Themixture was stirred for 2 h at room temperature, and then GPGK(Dde)LVFF dissolved in DIPEA was added. After stirring at room temperature for 18 h, the solventwas removed under vacuum, the solid was washed by centrifugation,three times with diethyl ether and two times with 0.01 N HCl. Theresidue was dried in vacuo. Pure compound 4  was obtained by preparative TLC. The Dde protectinggroup was removed from compound 4 by treatment with 2%hydrazine (1 mL), under stirring, at rt, for 30 min. After removalof hydrazine under vacuum, 20% TFA in CH2Cl2 (1 mL) was added to the solid, for removing the Boc groups. Themixture was stirred at room temperature for 2 h. The solvent was removedunder vacuum, and the solid was washed three times with diethyl etherby centrifugation, to give pure compound 5 after freezedrying . Sample identity was confirmed by MALDI-TOFmass spectrometry. Calculated mass for C83H112N14O13: 1512.8533; observed: 1536.0288 (M+ Na)+; 1551.9912 (M + K)+. 1H NMR δ 0.70 , 0.80 , 0.87 , 0.88 , 0.93 , 0.96 , 1.27 , 1.44–1.60 , 1.60–1.90 , 1.90–2.00, 2.00–2.10 , 2.13 and 2.24 , 2.80 , 2.80–3.20 , 3.20–3.43 , 3.71–3.93, 3.96 , 4.10  4.20–4.36, 4.36–4.61 , 6.34 , 6.38, 6.46 , 6.66 , 6.70 , 6.79 ,7.09–7.37 . 13C NMR : 10.5, 10.8 (q), 18.9,19.4, 21.8, 21.9 (q), 23.4 23.6, 24.0, 25.0 (d), 25.7, 27.4, 30.3,38.2, 38.5, 40.3, 40.8, 42.4, 44.2 (t), 54.2, 54.6, 55.9, 56.5, 61.1,62.2 (d), 74.7, 77.9 (t), 122.1, 123.2, 127.6, 127.7, 129.3, 129.4,130.0, 130.2 (d), 136.6, 136.7, 137.1, 138.0, 138.3 (s), 169.3, 171.9,172.1, 172.9, 173.4, 174.3, 175.1, 175.6 .To 20 mg of calixarene derivative 42 was dissolvedin TFA (1 mg/mL) and sonicated for 10 min. Then the TFA was removedunder a gentle stream of N2, 1 mL of HFIP was added, andthe mixture was dried under N2 stream to remove the remainingtrace of TFA (×2). Aβ42 was again dissolvedin 1 mL of HFIP and was frozen at −70 °C and then lyophilizedovernight. The same procedure was carried out in the presence of conjugate 5.Aβ2 at room temperature. The CD spectra were recorded forAβ42 (5 μM) monomer in the absence and presenceof 1 and 5 (5 and 25 μM). The lyophilizedsamples were dissolved in 50 μL of 10 mM NaOH and then dilutedwith 10 mM phosphate buffer pH 7.4 containing 100 mM NaCl to 2 mLto obtain a concentration 5 μM for Aβ42 aloneand the mixture of Aβ42 and conjugate 5 (1:1 and 1:5 molar ratio). A 1 cm path length quartz cuvette wasused to acquire the far-UV CD spectra (190–260 nm) at a scanspeed of 50 nm/min. There were 10 scans collected. The CD signal ofthe solution without Aβ42 was subtracted from thesample CD spectra. The measurements were performed in triplicate.CD spectra were acquiredusing a J-810 spectrometer  under a constant flow ofN42 alone and in the presence of the conjugates 1 or 5 in 1:1 or 1:5 molar ratio was dissolved in 10mM aq NaOH (30 μL). The samples were diluted (to 150 μL)with 60 μM ThT solution in 10 mM phosphate buffer at pH 7.4to obtain a final Aβ42 concentration of 20 μM.The samples were incubated a 37 °C in a 96-well plate. To minimizeevaporation effects the multiwell plate was sealed with a transparentheat-resistant plastic film. Readings were taken every 10 min, afterweak shaking for 10 s. The fluorescence intensity was monitored for67 h. The measurements were performed in triplicate. To minimize errorsduring sample preparation, we freeze dried the aliquots of monomerizedAβ42 and 5 directly into each well ofthe plate. The experimental data were fitted by using the equation:F0 is theinitial fluorescence emission and Fmax is the final increment of fluorescence emission; 1/k is the elongation rate constant, and t1/2 is the time at which the amplitude of ThT emission is 50% of the Fmax value. tlag isdefined as the intercept between the time axis and the tangent ofthe curve with slope k from the midpoint of the fittedsigmoidal curve; this parameter was calculated from the fitted parametersby using the following equation: tlag = t1/2 – 2k.ThT fluorescence kineticswere measured on a Flash Thermo Varioskan spectrofluorometer withexcitation and emission at 450 and 480 nm, respectively. AβThekinetic parameters are expressed as the mean (±SD) of three independentexperiments.42, conjugate 5, and Aβ42 in the presence of 5 was determined on samplesprepared at the same experimental conditions as CD analyses. Eachmeasurement was performed three times.DLS and ELS measurements were carried out on a ZetaSizerNanoZS90 Malvern instrument (UK), equipped with a 633 nm laser ata scattering angle of 90° and 25 °C temperature. The sizeof Aβ2. The measurements were carried out in tappingmode using a silicon Sn doped tip (resistivity of 0.01 Ω cmand purchased by Bruker TESPA). New cantilevers were used for eachmeasurement. Tip dimensions: thickness 4 μm, length 125 μm,width 40 μm. The stiffness was 40 N/m, and the tip was operatedto an oscillation frequency of 320 kHz. An aliquot with a volume of10 μL of sample was dispensed on a precleaned silicon flat substrateand dried. In order to perform a representative investigation of wholesamples, after deposition the substrates were not rinsed with Milli-Qwater (as commonly reported in literature). For each sample, variousareas on the sample were investigated and statistically relevant imageswere chosen.AFM analysis was performedwith a PSIAXE-150 system, acquiring images of 1 × 1 and 2 ×2 μm42 was dissolved in HFIP (hexafluoroisopropanol)to obtaina concentration of 2.2 × 10–4 M (Aβ42 stock solution). The GPGKLVFF peptide and compound 5 were dissolved in HFIP to obtain a stock solution of 1.1× 10–3 M for each. The Aβ42 sample and the mixtures of Aβ42/GPGKLVFF and Aβ42/5 at 1:5 ratios, were prepared from stock solutions to afinal concentration [Aβ42] = 5.5 × 10–6 M, [5] = [GPGKLVFF] = 27.5 × 10–6 M in Milli-Q water at physiological pH. For proteolysis experiments,25 × 10–6 g of trypsin enzyme was dissolvedin HCl 1 × 10–3 M [trypsin] = 2.14 × 10–5 M, and then an appropriate volume of the enzyme stocksolution was added to the Aβ42, Aβ42/GPGKLVFF, and Aβ42/5 samples to obtaina final enzyme/substrate ratio of 1:20 w/w. Solutions were incubatedat 37 °C, and then the digestion reactions were stopped after2 h by adding 1 μL 0.3% aqueous TFA. The samples were analyzedby using an ESI-MS Orbitrap Q-exactive system (Thermos Scientific).Each sample was introduced into the ESI source on 100 mm internaldiameter fused silica via a 500 mL syringe. Full MS scans in the m/z range 400–4000 were acquiredin the positive ion mode, spray voltage = 3.5 kV, capillary temperature= 300 °C; m/z range = 400–4000,S-lens RF level = 60 V, sheath gas = 7, resolving power: 70 000fwhm. The spectra, recorded as raw files, were imported in Qual Browser(Thermo Scientific) software for analysis. Averaged MS spectra wereimported into a freely available open-source software, mMass (http://www.mmass.org). Theoretical m/z values of Aβ42, GPGKLVFF, 5, the adducts Aβ42/GPGKLVFF and Aβ42/5, and the peptides resulting from in silicodigestion of Aβ42 were compared with the m/z values assigned to experimental massspectra. Peptides that matched successfully, within a tolerance of0.005 Da, were annotated.The lyophilizedAβl-glutamine at 37 °C, 5% CO2. Twoweeks before experiments, 5 × 103 cells were platedon 96-well plates in DMEM-F12 with 5% HI fetal calf serum. The percentageof serum was gradually decreased until it was 1% of the total. All-trans-retinoic acid (RA) (Sigma), 5 μM, was used topromote neuronal differentiation, and medium-containing RA was changedevery 3 days. Treatments with compound 5, KLVFF, andGPGKLVFF were performed on fully differentiated cells. After 24 htreatment, cultures were incubated with MTT (0.5 mg/mL) for 2 h at37 °C and then lysed with DMSO, and the formazan production wasevaluated in a plate reader through the absorbance at 570 nm.Theneuroblastoma (NB)cell line, SH-SY5Y, was maintained in DMEM-F12 supplemented with 10% heat inactivated (HI) fetal calf serum , 100 mg/mL penicillin and streptomycin ,and 2 mM 42 oligomerswere prepared as previously described54 from synthetic Aβ42 following a protocol for monomerization.An amount of 1 mg of Aβ42 was first dissolved in5 mM DMSO. A solution of 10 μM Aβ42 in ice-coldDMEM F-12 without Phenol Red was prepared and allowed to oligomerizefor 48 h at 4 °C according to the Lambert protocol55 with some modification as previously described.56 In order to evaluate the ability of compound 5 and the other appropriate controls , samplesof Aβ42 (10 μM) were incubated in the presenceor absence of each compound . After48 h incubation, Aβ/ligand compounds were applied to the differentiatedSH-SY5Y cells at the final concentration of 2 μM Aβ.Aβ42 with KLVFF peptide, GPGKLVFF, orcompound 5 with a molar ratio Aβ/peptide of 1:5.All the samples, including Aβ alone at the two concentrationsused, were incubated at 4 °C for 48 h to form Aβ oligomers.After incubation, the amount and size of Aβ aggregates weredetermined by Western blot analysis. A volume of 25 μL of eachunheated sample was loaded onto a precast Bis-Tris gel  with 2-morpholin-4-yl ethanesulfonic acid (MES).Samples were transferred onto a nitrocellulose membrane  by using a wet transfer unit Mini Blot Module(Life Technologies). Membranes were blocked in Odyssey blocking buffer(Li-COR Biosciences) and incubated at 4 °C overnight with mousemonoclonal anti-amyloid-β antibody against N-terminal 1–16peptide (1:1000) (Covance). A secondary goat anti-mouse antibody labeledwith IR dye 800 (1:25 000) was used at rt for 45 min. Hybridizationsignals were detected with the Odyssey CLx infrared imaging system.A stock aliquot of 5 mM Aβpreviously dissolved in DMSO was diluted in DMEM F-12 without PhenolRed, and two different samples at 20 μM and 50 μM concentrationswere prepared. From each solution, two sets of samples were obtainedby combining Aβ


The mutations G170R and I244T are the most common disease cause in primary hyperoxaluria type I (PH1). These mutations cause the misfolding of the AGT protein in the minor allele AGT-LM that contains the P11L polymorphism, which may affect the folding of the N-terminal segment (NTT-AGT). The NTT-AGT is phosphorylated at T9, although the role of this event in PH1 is unknown. In this work, phosphorylation of T9 was mimicked by introducing the T9E mutation in the NTT-AGT peptide and the full-length protein. The NTT-AGT conformational landscape was studied by circular dichroism, NMR, and statistical mechanical methods. Functional and stability effects on the full-length AGT protein were characterized by spectroscopic methods. The T9E and P11L mutations together reshaped the conformational landscape of the isolated NTT-AGT peptide by stabilizing ordered conformations. In the context of the full-length AGT protein, the T9E mutation had no effect on the overall AGT function or conformation, but enhanced aggregation of the minor allele (LM) protein and synergized with the mutations G170R and I244T. Our findings indicate that phosphorylation of T9 may affect the conformation of the NTT-AGT and synergize with PH1-causing mutations to promote aggregation in a genotype-specific manner. Phosphorylation should be considered a novel regulatory mechanism in PH1 pathogenesis. The native state ensemble of proteins contains folded, partially folded, and unfolded microstates populated according to their Gibbs free energy, which can be regarded as macroscopic substates . These dAGXT gene causing loss-of-function in the alanine:glyoxylate aminotransferase (AGT) enzyme ) were determined more accurately and clearly showed very little effect of the T9E and T9A mutations   . We then carried our PLP binding experiments by analyzing its binding kinetics under pseudo first-order conditions at 25 °C . This pr3 M·s−1) . The res 2.2 µM) A. As expagnitude ,47, likevariants C. HoweveTm between T9E or T9A mutant and control groups were: apo-T9E group: −0.2 ± 0.8 °C; holo-T9E group: 0.5 ± 0.7 °C; apo-T9A group: 0.3± 1.2 °C; holo-T9E group: 0.3 ± 0.6 °C; note that a negative value would indicate reduced stability upon T9E/T9A mutation). Thermal stability of AGT is largely increased upon binding of PLP ,48 Figu. InteresEnhanced protein aggregation is a common effect of PH1-causing mutations, particularly in the apo-AGT state ,31,48,49extent (scattering at 400 nm after a 90 min incubation) and maximal rate  of aggregation  MRE), we used Equation (1):Θobs was the ellipticity (in deg), l was the path length (in cm), and c was the concentration of peptides/AGT in g·mL−1. To calculate mean residue ellipticities ([Θ]MRE([TFE]) to Equation (2) [Θ]MRE,TFE was the mean residue ellipticity of the TFE-induced conformational transition , [Θ]MRE,buffer was the mean residue ellipticity of the peptides in buffer, m describes the cooperativity of the transition , Cm was concentration for half-transition (in M), and R was the ideal gas constant . To provide an accurate estimation of m- and Cm values, we simultaneously fitted the results from 200 and 225 nm using Equation (2). Using the linear extrapolation method, the folding free energy in the absence of TFE (ΔG) can be calculated using Equation (3):To estimate the apparent free energy change associated with TFE-induced conformational transitions of the NTT-AGT peptides, we used a two-state equilibrium model to fit the TFE-concentration ([TFE] in M)-dependent mean residue ellipticity MRE at 200 or 225 nm from 5 °C to 85 °C or 95 °C at a scan rate of 1.5 °C·min−1. Every 10 °C step, samples were thermostatized for 2 min and four scans were acquired and averaged at that temperature. Once a thermal scan concluded, the sample was cooled down to 5 °C, thermostatized for 5 min, and four scans were registered and averaged. In all cases, scans with buffer and no peptide were registered and subsequently subtracted.Thermal scans of the AGT-NTT peptides were carried out under the same conditions (peptide concentration and buffer) by acquisition of [1H-NMR spectra: A total of 48 scans were acquired with 16 K acquisition points for the homonuclear 1D-1H-NMR spectra of each isolated peptide at a concentration of 1.2 mM. Water signal was suppressed with the WATERGATE sequence [1D-sequence . The spe2O. Details on the experimental conditions and fitting of the resulting curves have been described elsewhere [D, for the residual proton water signal in a sample containing 100% D2O, in a 5-mm tube [Rh of 2.12 Å [Translational diffusion NMR (DOSY): The peptide concentrations in DOSY experiments was 100 µM, and 128 scans were acquired, where the gradient strength was varied linearly. Measurements of the translational self-diffusion were performed with the pulsed-gradient spin-echo sequence in the presence of 100% Dlsewhere . The gra-mm tube . The lenf 2.12 Å , was add1H-NMR spectra: Two-dimensional spectra of the four peptides in aqueous solution  or in the presence of deuterated TFE were acquired in each dimension in phase-sensitive mode by using the time-proportional phase incrementation technique [1H-NMR experiments. Standard TOCSY (with a mixing time of 80 ms) [per increment in the first dimension, and the residual water signal was removed by using the WATERGATE sequence [1H resonances were assigned by standard sequential assignment processes [α protons in random-coil regions were obtained from tabulated data, corrected by neighboring residue effects [2D-echnique  and a spf 80 ms)  and NOESf 80 ms)  were perf 80 ms)  was usedsequence . NOESY ssequence , with a rocesses . The che effects ,45,46.The NMR spectra were acquired at 10 °C on a Bruker Avance 500 MHz spectrometer , equipped with a triple resonance probe and z-pulse field gradients. Spectra were processed with Bruker TopSpin 2.1 . All NMR experiments with the four peptides were carried out in 100 mM sodium phosphate buffer (not corrected for isotope effects), pH 7.2. Spectra were calibrated with TSP by considering pH-dependent changes of its chemical shifts ; probe tN2, where N is the peptide length [−1 and an entropic penalty for fixing a residue in the native conformation of −16.5 J·mol−1·K−1 per residue for all residues, other than proline, glycine, and non-helical residues. All non-helical and glycine residues were provided an additional entropic penalty of −6.06 J·mol−1·K−1 per residue [The conformational features of the peptides are predicted by the exact-solution to the Wako-Saitô-Muñoz-Eaton (WSME) model that assumes an ensemble of e length ,69. In t residue , given tE. coli strains were transformed with these plasmids, and protein expression was induced by addition of 0.4 mM IPTG for 4–6 h at 4 °C. AGT proteins were purified from soluble extracts by metal affinity chromatography and subsequent size exclusion chromatography, as described in [280 = 47,000 M−1·cm−1 for the AGT monomer. Unless otherwise indicated, all subsequent experiments were carried out in the same buffer. Mutations to T9E and T9A were introduced into pCOLDII plasmids containing AGT WT, LM, LM-G170R, or LM-I244T  using stribed in ,32. Apo −1 purified AGT protein. The reaction mixture contained 10 mM glyoxylate, 100 mM L-alanine, and 150 µM PLP in 100 mM K-phosphate pH 8. The reaction time was set to 2 min and the reaction quenched by using trichloroacetic acid . Samples were clarified by centrifugation at 21,000 g for 10 min at 4 °C, and supernatants were stored at −20 °C. Pyruvate formed in these reactions was determined using lactate dehydrogenase and NADH 0.2 mM at 37 °C. The reaction was monitored as the change in A340 nm in TRIS-HCl 1 M pH 8 using quartz cuvettes in an Agilent HP8453 spectrophotometer at 25 °C. These amounts of pyruvate were determined using calibration curves of pyruvate under the same conditions. For each variant, we carried out at least five different replicas, and the results were presented as mean ± s.d. AGT-specific activity was measured at 37 °C, typically using 2.5 (32 nM) to 4  µg·ml®6, Sartorius), and the spectra of filtrates was acquired under similar conditions to those used with AGT proteins. CD measurements were performed at 25 °C in a Jasco J-715 spectropolarimeter equipped with a Peltier temperature controller. AGT samples for CD measurements in the near UV-visible range were prepared as described above for absorption measurements. Spectra were recorded at a scan rate of 100 nm·min−1 with a 2 nm bandwidth, and five scans were registered and averaged using 5 mm path-length cuvettes, and appropriate blanks without protein were registered and subtracted. For far-UV CD measurements, AGT proteins were prepared at 5 µM (in subunit) in K-phosphate 20 mM pH 7.4, and spectra were recorded at a scan rate of 100 nm·min−1 with a 1 nm bandwidth, and six scans were registered and averaged using 1 mm path-length cuvettes, and appropriate blanks without protein were registered and subtracted. Absorption and CD spectra were recorded from two different preparations of each AGT variant (two independent purifications) and averaged. All CD data were normalized for protein concentration using Equation (2). Dynamic light scattering (DLS) was measured using a DynaPro MSX instrument (Wyatt) in a 1.5 mm path-length cuvette and using 10 µM (in subunit) AGT proteins, as purified. Twenty-five spectra were acquired at 25 °C for each DLS analysis, averaged, and used to determine the hydrodynamic radius assuming spherical scattering particles . Radii were expressed as mean ± s.d. from 4–6 replicates.All spectroscopic measurements were carried out in Na-HEPES 20 mM NaCl 200 mM pH 7.4, unless otherwise indicated. UV-visible absorption spectra were collected in an Agilent HP8453 diode-array spectrophotometer at 25 °C using 1 cm path length cuvettes and 20 µM (in subunit) AGT proteins, as purified. To trigger the formation of PMP, a final concentration of 0.2 M L-alanine was added, and the reaction was allowed to proceed for at least 10 min at 25 °C before spectra collection. To determine whether the PMP formed was released upon addition of L-alanine, these reactions were filtered using 30 kDa cut-off concentrators  were simply calculated, considering that these are equal to the inverse of Ka (=1/Kd). PLP binding to apo proteins was measured under pseudo first-order kinetic conditions. PLP (5–100 μM) was always in a large excess, compared to apo-AGT . Protein samples were thermostatized in 1 cm quartz-cuvettes at 25 °C for five minutes, and PLP was added to an appropriate final concentration and mixed manually . Time-dependent emission fluorescence was acquired in a Cary Eclipse spectrofluorimeter , using an excitation wavelength of 280 nm and an emission wavelength of 340 nm (5 nm slits) at 25 °C. Kinetic traces were fitted to a single exponential function to provide the observed rate constant −1. Upon subtraction of pre- and post-transition linear baselines, the apparent denaturation temperature (Tm) was determined as the temperature at which half of the denaturation signal was achieved. Values reported are mean ± s.d. from 4 replicas for each protein variant. Thermal stability of AGT proteins (2 μM in protein subunit) was evaluated by following the changes in fluorescence emission intensity  using a Cary Eclipse spectrofluorimeter . Holo-AGT samples contained proteins, as purified with 100 μM PLP, while apo proteins were prepared from apo protein stored solutions. All protein solutions were prepared in Na-HEPES 20 mM NaCl 200 mM pH 7.4 and placed in 1 cm path-length cuvettes. After 5 min of incubation at 20 °C, thermal scans were carried out from this temperature up to 70–95 °C at a scan rate of 2 °C·min400) was then monitored for 90 min. Control cuvettes contained only buffer. From these transients, two different parameters were obtained: the maximal A400 after 90′ and the maximal rate (from the first derivative of A400 vs. time). Values reported are mean ± s.d. from 4 replicas for each protein variant. Aggregation kinetics were measured in an Agilent 8453 diode-array spectrophotometer at 37 °C using 1 cm path length plastic cuvettes and 2 µM (in protein subunit) AGT proteins. A total of 980 µL of Na-HEPES 20 mM NaCl 200 mM pH 7.4 was placed in the cuvettes and thermostatized for 10 min at 37 °C. Then, aggregation measurements were initiated by the addition of 20 µL of 50 µM of holo or apo proteins and rapid mixing with a glass pipette. The apparent absorbance at 400 nm (A


RS1 gene on Xp 22.13 encodes retinoschisin which is known to directly interact with the retinal Na/K-ATPase at the photoreceptor inner segments. Pathologic mutations in RS1 cause X-linked juvenile retinoschisis (XLRS), a hereditary retinal dystrophy in young males. To further delineate the retinoschisin-Na/K-ATPase complex, co-immunoprecipitation was performed with porcine and murine retinal lysates targeting the ATP1A3 subunit. This identified the voltage-gated potassium (Kv) channel subunits Kv2.1 and Kv8.2 as direct interaction partners of the retinal Na/K-ATPase. Colocalization of the individual components of the complex was demonstrated at the membrane of photoreceptor inner segments. We further show that retinoschisin-deficiency, a frequent consequence of molecular pathology in XLRS, causes mislocalization of the macromolecular complex during postnatal retinal development with a simultaneous reduction of Kv2.1 and Kv8.2 protein expression, while the level of retinal Na/K-ATPase expression remains unaffected. Patch-clamp analysis revealed no effect of retinoschisin-deficiency on Kv channel mediated potassium ion currents in vitro. Together, our data suggest that Kv2.1 and Kv8.2 together with retinoschisin and the retinal Na/K-ATPase are integral parts of a macromolecular complex at the photoreceptor inner segments. Defective compartmentalization of this complex due to retinoschisin-deficiency may be a crucial step in initial XLRS pathogenesis.The The online version contains supplementary material available at 10.1007/s00018-022-04409-9. RS1 gene, which is specifically expressed in photoreceptors and bipolar cells of the retina, as well as pinealocytes of the pineal gland [X-linked juvenile retinoschisis  is a hereditary retinal dystrophy affecting males with an estimated prevalence between 1:5.000 and 1:20.000. Characteristic features of XLRS are splitting of the inner retinal layers, and defective signal transmission from photoreceptors to bipolar cells [al gland  were shoRS1 gene encodes a secreted protein termed retinoschisin which is anchored to the retinal plasma membrane upon binding to the retinal Na/K-ATPase [The K-ATPase , 7. Na/KK-ATPase : a catalK-ATPase , 10. TheK-ATPase . In the K-ATPase , and thuK-ATPase . They arK-ATPase . SpecifiK-ATPase  IsoformsK-ATPase .To further elucidate the molecular processes underlying XLRS pathogenesis, we aimed to identify additional components of the retinoschisin-Na/K-ATPase complex and explored the consequences of retinoschisin-deficiency on the integrity of such a complex. We performed co-immunoprecipitation analyses targeting the ATP1A3 isoform of the retinal Na/K-ATPase in porcine and murine retinal lysates followed by an in depth-characterization of newly identified interaction partners, namely the voltage-gated potassium (Kv) channel subunits Kv2.1 and Kv8.2.−/y (Rs1tm1Web) mice were kept on a C57BL/6 J background for more than 40 generations. Mice were housed under specific pathogen-free barrier conditions at the Central Animal Facility of the University of Regensburg, in strict compliance with NIH guidelines. Mice were sacrificed by cervical dislocation at different postnatal days (P), specifically at P4, P7, P10, P14, P18, P21, or P30. Porcine eyes were obtained fresh from a local slaughterhouse.Rs1h, ATCC; LGC Standards GmbH, Wesel, Germany) and Hek293 cells  were cultivated as described by Biasella and colleagues [Y-79 (RRID:CVCL_1893lleagues . For theKCNB1 (NM_004975.4) and KCNV2 (NM_133497) were amplified from cDNA of Y-79  and cloned into the KpnI/NotI site of the pcDNA3.1 vector . Generation of expression constructs for untagged retinoschisin (NM_000330.4) is described by Friedrich and colleagues [The coding sequences of lleagues , for thelleagues , and forlleagues .TransIT-LTI Transfection Reagent (Thermo Fisher Scientific). For heterologous expression of retinoschisin, Hek293 cells were transfected using the calcium-phosphate method [For heterologous expression of Kv channels (Kv2.1/Kv8.2) or the retinal Na/K-ATPase (ATP1A3/ATP1B2), Hek293 cells were transfected with Mirus Bio e method .48 h after transfection, cell-surface expression of heterologously expressed proteins was analyzed using the Pierce™ Cell-Surface Protein Isolation Kit (Thermo Fisher Scientific). According to the manufacturer’s instructions, cell-surface proteins were biotinylated, purified and subsequently analyzed by western blot analysis.Retinoschisin binding to Hek293 cells heterologously expressing the retinal Na/K-ATPase, Kv2.1, Kv8.2, or Kv2.1 plus Kv8.2 was performed as described , 17, 19,SDS-PAGE and western blot analysis were performed as previously described in , 20. DetProtein A Sepharose beads  coupled to antibodies, were prepared as follows: 50 µl of the beads were washed three times with 1 ml of phosphate-buffered saline (PBS) and spun down for 2 min at 2000 rpm at 4 °C. After discarding the supernatant, beads were conjugated with the respective antibodies, beads and antibody solution (2 µg in 100 µl PBS) were mixed and rotated at 4 °C for 1 h followed by centrifugation for 2 min at 2000 rpm at 4 °C. Subsequently, the supernatant was discarded, and antibody-coupled beads were mixed with the input.Retinal lysates were prepared as follows: for each co-immunoprecipitation experiment, 10 mg of porcine retinae was dissolved in 3 ml of 10 mM Cholamidopropyl-dimethylammonio-1-propansulfonat (CHAPS) in Tris-buffered saline (TBS) followed by sonification time at 40% for of 45 s. Alternatively, four retinae of wildtype mice (P18) were homogenized in 1 ml of 10 mM CHAPS in TBS and sonicated for 10 s at 40%. The suspensions were rotated at 4 °C for 1 h and cell debris was removed by centrifugation for 20 min at 13,000 rpm and 4 °C. The supernatant containing the retinal lysate was collected and used as “input”.Input (3 ml of porcine lysate or 1 ml of murine lysate) was added to the antibody-coupled beads. After overnight rotation at 4 °C, the beads were spun down for 2 min at 2000 rpm and 4 °C. 50 µl of the supernatant was collected as “flow through” fraction and the rest was discarded. To remove unbound protein, beads were washed five times with 1 ml 10 mM CHAPS in TBS (centrifugation as described at each step) and 100 µl of the last wash fraction (“wash”) was collected. Precipitated proteins were eluted with 1 × Laemmli-buffer (Laemmli 1970), and input, flow through, and wash fractions were mixed with 5 × Laemmli-buffer. Samples were denatured at 93 °C for 10 min and subjected to Coomassie Blue staining  or western blot analysis (murine lysates). In co-immunoprecipitation experiments the flow through fraction was frequently similar in intensity to the input. This suggests an excess of protein exceeding the binding capacity of the beads and leading to a minor decrease of the total protein amount after co-immunoprecipitation (and thus similar flow through intensities). Moreover, immunolabelling signals in the precipitate fractions were frequently weak, especially for ATP1A3, Kv2.1, and Kv8.2. Due to inherent properties of membrane proteins , co-immu4HCO3 and lyophilized. After a reduction/alkylation treatment and additional washing steps, proteins were in gel digested with trypsin  overnight at 37 °C. The resulting peptides were sequentially extracted with 50 mM NH4HCO3 and 50 mM NH4HCO3 in 50% acetonitrile. After lyophilization, peptides were reconstituted in 20 µl 1% TFA and separated by reversed-phase chromatography. An UltiMate 3000 RSLCnano System  equipped with a C18 Acclaim Pepmap100 preconcentration column  and an Acclaim Pepmap100 C18 nano column  was operated at flow rate of 300 nl/min and a 60 min linear gradient of 4% to 40% acetonitrile in 0.1% formic acid. The LC was online-coupled to a maXis plus UHR-QTOF System  via a CaptiveSpray nanoflow electrospray source. Acquisition of MS/MS spectra after collision-induced dissociation (CID) fragmentation was performed in data-dependent mode at a resolution of 60,000. The precursor scan rate was 2 Hz processing a mass range between m/z 175 and m/z 2000. A dynamic method with a fixed cycle time of 3 s was applied via the Compass 1.7 acquisition and processing software .Proteins were separated on a precast 4–12% NUPAGE Bis–Tris gel (Invitrogen) and stained with Coomassie Blue. Gel lanes were cut into stripes, washed with 50 mM NHSus scrofa database with the following parameters: enzyme specificity trypsin with two missed cleavages and cleavage before proline allowed, precursor tolerance 0.02 Da and MS/MS tolerance 0.04 Da. Deamidation of asparagine and glutamine, oxidation of methionine, Nt-Acetylation, carbamidomethylation or propionamide modification of cysteine were set as variable modifications. Calculation of iBAQ values was activated and protein and peptide identifications were filtered at a false discovery rate (FDR) < 1%. For complex stoichiometry estimation iBAQ value ratios were calculated using MS Excel (Version 2018).MaxQuant Version 2.0.3.0  was usedRNA was isolated from murine retinae of wildtype and retinoschisin-deficient mice using the PureLink™ RNA Micro Kit (Invitrogen), according to the manufacturers’ protocols. One microgram of total RNA was transcribed into cDNA using Random Hexamer Primers (Thermo Fisher Scientific) and the RevertAid M‐MuLV Reverse Transcriptase  according to the manufacturer's instructions. Quantitative real‐time PCR was performed in technical triplicates using KiCqStart® probe assays from Sigma Aldrich (Supplementary Table S5) and the QuantStudio5 System (Thermo Fisher Scientific). Results were analyzed by applying the ΔΔCt method for relative quantification .Glass cover slips (12 mm) were coated with Poly-L-Lysin for 1 h at 37 °C. Hek293 cells were seeded on coated cover slips, transfected with the different expression constructs, and cultured for 24 h. Immunocytochemical analysis was performed as described by Brandl and colleagues [Murine eyes  were enucleated, fixed, embedded, cryosectioned, and immunolabeled as described by Friedrich and colleagues . AntibodFor each replicate, the two retinae of a single mouse  were voltage-clamped at a holding potential of − 60 mV with voltage steps between − 60 and 80 mV in 10 mV increments. The whole-cell voltage-clamp recordings were performed by using an EPC-10 USB amplifier on an Olympus IX73 microscope  and the Patchmaster Software . To test whether the measured outward current is generated by potassium channels, KCl in intracellular and extracellular solutions was replaced by CsCl or the specific Kv2 channel inhibitor citalopram  was added to the extracellular solution.Glass cover slips (18 mm) were coated with concanavalin-A (0.5% in 1 M NaCl) for 2 h at 37 °C and then rinsed with distilled water. Y-79 cells were seeded on the coated cover slips and cultured for 24 h in medium containing purified retinoschisin (1 µg/ml) or the same volume of control eluate. Electrophysiological measurements of potassium channel currents were performed as described by Gomez M del P and colleagues , with slU test . Data following a Gaussian distribution were analyzed using Student’s t-test . Statistical analyses were performed using the “XLSTAT add-in” software.Normality of the data was assessed by the Shapiro–Wilk normality test. Data not following a Gaussian distribution were analyzed using Mann–Whitney-To identify new interaction partners of the retinal Na/K-ATPase, we initially performed co-immunoprecipitation experiments with porcine retinal lysates targeting ATP1A3, followed by mass spectrometric analysis of precipitated proteins. Co-immunoprecipitation experiments with anti-6*His-tag antibodies were included as a negative control. A comprehensive list of proteins detected by mass spectrometry in the precipitate fractions of both experimental approaches  is given in Supplementary Table S1 and S2. ATP1A3 revealed the highest abundance reflected by an intensity-based absolute quantification (iBAQ) value. ATP1B2 and retinoschisin were also detected albeit at lower levels (Supplementary Table S1). This demonstrates the capacity of the co-immunoprecipitation approach to precipitate ATP1A3 interacting proteins, like ATP1B2, and proteins attached to ATP1A3 binding proteins, like the ATP1B2 binding partner retinoschisin.A candidate prioritization was done to select suitable proteins for further analysis. From a list of proteins exclusively detected or at least fivefold enriched in the ATP1A3-co-immunoprecipitation compared to control for all three replicates (Supplementary Table S1), candidates of interest were selected. To further refine the potential list of candidates, an extensive literature search was done focusing on proteins of the retina localizing to the inner segment (IS) of the photoreceptors, close to the retinal Na/K-ATPase). This focused our interest on Kv channel subunits Kv2.1 and Kv8.2, as both proteins have been localized to the photoreceptor IS membrane –29 and aStoichiometry of the ATP1A3 complex protein components chosen for further analyses was determined by calculating the ratios of the intensity based absolute quantification (iBAQ) values of ATP1A3 and the respective candidate proteins. The data revealed sub-stoichiometric amounts of Kv2.1 and Kv8.2 compared to ATP1A3 in the precipitate fractions. Detailed information on the stoichiometry, iBAQ values and sequence coverage of the ATP1A3 interaction partners ATP1B2, retinoschisin, Kv2.1, and Kv8.2 is given in Supplementary Table S2.To confirm the interaction of ATP1A3 and the Kv channel subunits Kv2.1 and Kv8.2 in an independent approach, we performed co-immunoprecipitation with antibodies against Atp1a3 in murine retinal lysates followed by western blot analysis. Again, Kv2.1 and Kv8.2 were precipitated with Atp1a3, together with Atp1b2 and retinoschisin localization of the retinal Na/K-ATPase and Kv channel subunits Kv2.1 and Kv8.2 at the photoreceptor IS via immunohistochemistry of murine retinal cryosections. Consistent with the current literature –29, 38, The localization of the complex partners was additionally analyzed in Atp1b2-deficient mice. These mice develop normally but display motor coordination deficits and retinal photoreceptor degeneration starting around postnatal day (P9) . In Atp1To gain insight into the assembly of the Na/K-ATPase macromolecular complex, we examined whether retinoschisin, presenting multiple ligand binding sites in its hexadecameric conformation , 41, canEarlier analyses revealed that retinoschisin-deficiency affects the correct localization of the retinal Na/K-ATPase during murine retinal development starting at around P14 . Here, wP < 0.05))    of approximately 50% of Kv2.1 protein expression from P14 onward  from P18 onward , and Kcnv2 (encoding Kv8.2) in murine retinoschisin-deficient and wildtype retinae was compared at P14, P18, and P21. Quantitative RT-PCR revealed no statistically significant differences with only one exception . At P14, retinoschisin-deficiency induced a slight but statistically significant increase in the mRNA expression of Atp1b2. Nevertheless, this effect was not reflected in differences in protein expression of Atp1b2.In a next step, we clarified whether the spatial redistribution of Kv2.1 and Kv8.2 due to retinoschisin-deficiency is accompanied by changes in protein expression. While western blot analysis revealed no differences in the total protein amount of Atp1a3 and Atp1b2 between retinal lysates from wildtype and retinoschisin-deficient mice at all developmental stages analyzed Fig. a–c, reti+ channel blocker cesium chloride . As shown in Fig. In mice, the permanent potassium outward current in photoreceptors arises from continuously active Kv2.1 and Kv8.2 channels . To inveTo evaluate an effect of retinoschisin on the Kv channel activity, we performed voltage-clamp recordings on Y-79 cells incubated with or without recombinant retinoschisin. Cells were incubated with control eluate and purified retinoschisin for 24 h Fig. h and i, RS1 gene linked to XLRS pathogenesis regularly result in an extracellular deficiency of the encoded protein retinoschisin regardless of the specific type of mutation [The majority of pathologic mutations in the mutation . To elucmutation . In addimutation  and galamutation  were promutation , 49. Evemutation . Subsequmutation . Here, wwww.proteinatlas.org/humanproteome/tissue/retina [By co-immunoprecipitation, we reproducibly demonstrate a physical interaction of subunit ATP1A3 of the retinal Na/K-ATPase complex with Kv channel subunits Kv2.1 and Kv8.2. Of note, iBAQ values of the complex partners reveal sub-stoichiometric amounts of Kv2.1 and Kv8.2 compared to ATP1A3 in the precipitate fractions. This may be explained by the fact that ATP1A3 shows a much more widespread distribution along the different retinal cell types/retinal layers than Kv2.1 and Kv8.2. Specifically, immunohistochemical and transcriptome analyses revealed retinal Kv2.1 and Kv8.2 protein expression predominantly in photoreceptors (e/retina ) with a e/retina , 50, 51,e/retina . In conte/retina , 53–55. In the co-immunoprecipitation analysis targeting ATP1A3, we failed to detect Cav1.3 or Cav1.4, both putative retinoschisin interaction partners as suggested by Shi and colleagues , 49. ThiThe physical interaction of the retinal Na/K-ATPase complex with Kv channel subunits Kv2.1 and Kv8.2 was corroborated by our immunohistological co-localization studies of both Kv channels and the retinal Na/K-ATPase showing similar line-scan profiles of Atp1b2 and Kv2.1 in the IS membrane. Retinoschisin binding assays revealed that retinoschisin does not directly bind to Kv channels, thus excluding Kv channels to be linked to the retinal Na/K-ATPase via an interaction with retinoschisin. Retinoschisin-deficiency, known to result in XLRS pathology, causes an increasing mislocalization of the retinal Na/K-ATPase and the Kv channel subunits Kv2.1 and Kv8.2 during postnatal retinal development of the XLRS mouse retina. In addition, it is accompanied by a decrease of Kv2.1 and Kv8.2 protein beyond P14, whereas protein levels of the retinal Na/K-ATPase appear to be unaffected by retinoschisin deficiency. Of note, we could not observe an effect of retinoschisin on the Kv channel mediated potassium ion currents as analyzed in Y-79 cells. It remains to be shown to what extend the pathological findings of each member of the retinoschisin-retinal Na/K-ATPase-Kv channel complex contributes to initial or advanced disease development.Consistent with our current findings, Kv2.1 and Kv8.2 were shown earlier to be highly expressed in photoreceptor IS, but absent or poorly expressed in other retinal layers , 27. ThiSimilar to retinoschisin-deficient mice –58, receKcnb1 and Kcnv2 as well as for Atp1a3 and Atp1b2 in wildtype and retinoschisin-deficient retinae of P12 and P21 old mice [RT-PCR analysis revealed no changes in mRNA gene expression as the cause for altered protein levels of Kv2.1 and Kv8.2. This is in line with a previous study by Vijayasarathy and colleagues, who used microarray-based genome-wide expression profiling and observed no mRNA expression differences for old mice . Accordiold mice . The staold mice –62. RetiOur findings in the patch clamp analyses reveal no effect of retinoschisin binding on Kv channel activity in Y-79 cells. This is in line with the incapability of retinoschisin to directly bind to heterologously expressed Kv2.1/Kv8.2, as demonstrated in this study. Moreover, it disagrees with an immediate effect of retinoschisin binding to the macromolecular Na/K-ATPase complex on Kv channel activity, e.g. in a similar way as cardiotonic steroid binding to the Na/K-ATPase transactivates a variety of Na/K-ATPase associated proteins such as SRC, the IP3-receptor or the EGF-receptor, via structural alterations or trans-phosphorylation .Still, retinoschisin-deficiency could have a serious impact on Kv channel activity in vivo, putatively not by the immediate consequences of a disrupted complex binding, but by the observed negative effect on Kv channel localization and absolute channel density on IS membranes.KCNB1 encoding the Kv2.1 subunit, have been identified in patients with different neurodevelopmental disorders like epilepsy or autism [2+ levels due to elevated Ca2+ influx through cone cyclic nucleotide-gated (CNG) channels which ultimately causes rod degeneration [KCNV2, encoding Kv8.2, cause the retinal condition cone dystrophy with supernormal rod response (CDSSR) [Aberrant functionality of Kv channels or the Na/K-ATPase have been implicated in various pathological events before. It was reported that Na/K-ATPase binding to AnkB in cardiomyocytes controls the ion homeostasis via regulating the NCX activity in a local domain. Disruption of this interaction resulted in increased calcium sparks and waves, a possible mechanism for arrhythmogenesis in the AnkB syndrome . Also, pr autism . Furtherr autism , as wellneration . In the neration . Finally (CDSSR) .Taken together, our data suggest that retinoschisin may act as a crucial interaction partner of an emerging macromolecular complex at the IS of the mammalian photoreceptors by regulating the distribution and stability of the complex and its individual partners. An alteration in the spatial distribution and, consequently, the function of the complex may contribute to clinical symptoms in XLRS. Accordingly, it may be sensible to explore alternative ways to correct the secondary deficits of retinoschisin-deficiency as potential treatment options for XLRS.Supplementary file1 (docx 9852 KB)Supplementary file2 (pdf 877 KB)Supplementary file3 (XLSX 996 KB)Supplementary file4 (DOCX 16 KB)Supplementary file5 (DOCX 14 KB)Supplementary file6 (DOCX 14 KB)Supplementary file7 (DOCX 14 KB)Below is the link to the electronic supplementary material.


The treatment options for CTCL, in particular in advanced stages, are limited. One of the emerging therapies for CTCL is treatment with histone deacetylase (HDAC) inhibitors. We recently discovered an evolutionarily conserved crosstalk between HDAC1, one of the targets of HDAC inhibitors, and the histone methyltransferase DOT1L. HDAC1 negatively regulates DOT1L activity in yeast, mouse thymocytes, and mouse thymic lymphoma. Here we studied the functional relationship between HDAC inhibitors and DOT1L in two human CTCL cell lines, specifically addressing the question whether the crosstalk between DOT1L and HDAC1 observed in mouse T cells plays a role in the therapeutic effect of clinically relevant broad-acting HDAC inhibitors in the treatment of human CTCL. We confirmed that human CTCL cell lines were sensitive to treatment with pan-HDAC inhibitors. In contrast, the cell lines were not sensitive to DOT1L inhibitors. Combining both types of inhibitors did neither enhance nor suppress the inhibitory effect of HDAC inhibitors on CTCL cells. Thus our  The most common types are mycosis fungoides (MF) and Sézary syndrome (SS) that commonly originate from the CD4 lineage . MF is c lineage . SS pres lineage . Treatme lineage .One of the emerging systemic therapies that show promising results is treatment with histone deacetylase (HDAC) inhibitors . HDACs cDespite the clinical interest, the response towards HDAC inhibitors varies between patients and between different HDAC inhibitors . One potHdac1 knock-out mice also show increased H3K79me and this coincides with increased sensitivity towards DOT1L inhibitors  and DOT1-Like Histone Lysine Methyltransferase (DOT1L) . We recehibitors . Cryo-elhibitors .These observations lead to the question whether the observed crosstalk between DOT1L and HDAC1 in mouse T cells plays a role in the therapeutic effect of pan-HDAC inhibition in human CTCL. Predicting how HDAC1-DOT1L crosstalk affects CTCL is difficult because the role of HDACs in cancer is known to be context dependent. In mouse thymic lymphoma, HDAC1 deletion results in oncogenic transformation , whereas2 conditions.The human CTCL cell line Hut-78 (ATCC number TIB-161) and SeAx are derived from the peripheral blood of patients with SS . The hum2 conditions. After 4 hours, fluorescence was measured on the EnVision Multilabel Reader (Perkin Elmer). In order to calculate cell viability, values for fluorescence intensity of each condition were normalized against those of untreated cells. Each biological replicate represents an average of three technical replicates of an independent experiment.The HDAC inhibitors Panobinostat (Sigma) and Vorinostat (Sigma) and the DOT1L inhibitors Pinometostat (EPZ-5676)  and SGC-0946 , and DMSO (Sigma) were diluted in culture medium. First, the inhibitors were diluted in culture medium to a stock concentration that was twice as high as the highest concentration tested. The stock concentration was further diluted to obtain to the desired range of concentrations. Twenty-thousand cells were plated per well in a flat-bottom culture-treated 96 wells plate, in 50 µl culture medium and 50 µl culture medium with inhibitor was added. Four hours before measuring cell viability, Cell Titer Blue (Promega) was added to the wells and cells were incubated in the dark at 37°C under 5% CO2) containing 1:50 Annexin V-APC and 1:50 DAPI and subsequently incubated for 15 min at room temperature (shielded from light). After incubation the stained cells were transferred to tubes, the suspension was diluted by addition of 200 µL binding buffer and briefly kept on ice. Flow cytometry acquisition was subsequently performed using an LSR Fortessa (BD Biosciences) and the FACSDiva software v8.0.2 (BD Biosciences). Results were analyzed in FlowJo v10.5.3 (BD Biosciences).Viability, early and late apoptosis was measured by Annexin V-APC (Biolegend) and DAPI  staining. Staining was performed in 96-wells V-bottom plates and was previously optimized and titrated. Approximately 30,000–100,000 cells were washed in PBS, resuspended in 50 µL 1x binding buffer , one million cells (24-h incubation) and 0.5 million cells (72-h incubation) in 3 ml medium supplemented with 25 µM Pinometostat , 0.25 µM or 1 µM Vorinostat (Sigma), or with 0.025% DMSO (Sigma) as a vehicle control. Cells were washed with ice-cold PBS and pellets were snap-frozen in liquid nitrogen and stored at −80°C. Cells were lysed in ice cold RIPA buffer (25 mM Tris (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, 0.1% SDS) supplemented with complete protease inhibitor (Roche) and 5 times sheared over a 25G needle (BD). Subsequently, samples were sonicated in Eppendorf tubes for 5 min with a 30 s interval  using a Bioruptor Pico sonicator (Diagenode). Lysates were centrifuged for 2 min at 16,000x g to remove cellular debris. The supernatant was stored for use at −20°C for later use. The protein concentration of the lysates was determined using the DC protein assay (BioRad). Samples were boiled at 95°C for 5 min in 5x SDS-sample buffer  and separated on 16% polyacrylamide gels or NuPAGE 4–12% Bis-Tris gels (Invitrogen). Separated proteins were transferred on a 0.2 μm nitrocellulose membrane for 1 h at 1A (histone blots) or a 0.45 μm nitrocellulose membrane for 4 h at 1 A or overnight at 0.2 A (DOT1L blots). Membranes were blocked using 5% Nutrilon (Nutricia) in PBS for 30 min. Subsequently, membranes were incubated overnight at 4°C with the primary antibodies diluted in Tris-buffered saline containing 0.05% Tween-20 (TBST) and 2% Nutrilon: anti-H3K79me1 (RRID: AB_2631105) (1:1000), anti-H3K79me2  (1:1000), anti-H3K9ac  (1:2500), anti-H3K9ac  (1:1000), anti-H4Ac pan  (1:2000), anti-H2BK120ub1  (1:1000) DOT1L  (1:500), anti-α-Tubulin  (1:10000) or anti-H3  (1:5000). After incubation, the membranes were washed three times with TBST and incubated with secondary antibody for 45 min in TBST with 2% Nutrilon. Secondary antibodies used  were IRDye 800CW goat anti-Mouse IgG (0.5 mg) 926-32210 Li-COR (RRID:AB_621842), IRDye 800CW goat anti-Rabbit IgG (0.5 mg) 926-32211 Li-COR (RRID:AB_621843), IRDye 680RD goat anti-Mouse IgG (0.5 mg) 925-68070 Li-COR (RRID:AB_2651128), IRDye 680RD goat anti-Rabbit IgG (0.5 mg) 925-68071 Li-COR (RRID AB_2721181). Finally, the membranes were washed three times with TBST and once in PBS for subsequent scanning using a LI-COR Odyssey IR Imager (LI-COR Biosciences). Some of the blots were stripped and reprobed. Antibody stripping was performed by 15 min incubation in MiliQ, followed by 3 h incubation in 0.2 M NaOH and 15 min incubation in MiliQ. Stripping was confirmed by absence of signal on the scans after incubation with secondary antibody only. Stripped blots were subsequently stained with primary and secondary antibodies as described above.For the western blots in All statistical analyses were done using GraphPad Prism (version 9.4.1). Cell viability curves and half maximal effective concentration (EC50) values were determined using the ‘log (against) vs response–Find ECanything’ function, with top constraint to one and bottom constraint to 0, interpolation confidence interval: 95%, and untreated condition was set at -13 (Log10) in order to fit the line. Unpaired student’s t-test was used to determine difference between two different groups.To address the role of DOT1L in the therapeutic effect of HDAC inhibitors, we here studied single and combination treatments of two CTCL cell lines with the HDAC Class I, II and IV inhibitors Vorinostat (SAHA) and Panobinostat, both of which show promising results in CTCL treatment , and theWe first determined the effect of HDAC inhibitors on global levels of H3K79me. Cells were treated with two different concentrations of Vorinostat and H3K79me1/2 and H3K9ac and H4ac levels were assessed at different time points. Treatment with this HDAC inhibitor showed an increase in H3K9ac and H4 acetylation, serving as a proxy for global histone acetylation, confirming the reduction in HDAC activity . This efTo further investigate possible downstream effects of HDAC inhibitors that might affect DOT1L activity in CTCL cells, we looked at the best-known upstream regulator of H3K79 methylation. The activity of DOT1L towards H3K79 on the surface of the nucleosome is known to be enhanced by mono-ubiquitination of histone H2BK120 (H2BK120ub1) . Our pre50 levels obtained  by which they affect cell viability of CTCL cells is not completely known, although it has been shown that combined treatment of an HDAC inhibitor with a demethylating agent led to re-expression of a tumor suppressor gene and induction of apoptosis in CTCL cell lines . HDAC inresponse . In thisantation . Further in mice . The rolntiation . Inhibitpression . Given tpression  it would


E)-2-(pyridin-4-yl)vinyl) anthracene (BP4VA) are studied by means of surface-enhanced Raman scattering (SERS) spectroscopy and vibronic theory of resonance Raman spectroscopy. The SERS spectra recorded in an electrochemical cell with a silver working electrode have been interpreted on the basis of resonance Raman vibronic theory assisted by DFT calculations. It is demonstrated that the adsorbate–metal interaction occurs through the nitrogen atom of the pyridyl moiety. Concerning the electron donor–acceptor properties of the adsorbate, it is shown that the charge transfer excited states of BP4VA are not optically active, in contrast, an internal transition to an excited state of BP4VA, which is localized in the anthracene framework, is strongly allowed. The charge transfer states will be populated by an ultrafast non-radiative process, that is, internal conversion. Thus, irradiation of BP4VA interacting with an appropriate surface creates an effective charge separation.The electron donor–acceptor properties of 9,10-bis(( Surface enhanced Raman spectroscopy in conjunction with quantum chemistry is a valuable tool for characterization of organic functional materials. E)-2-(pyridin-4-yl)vinyl) anthracene  and theory are valuable tools to explore such donor–acceptor electron features. Thus, the present work focuses on the analysis of the surface-enhanced Raman scattering (SERS) spectra6,7 of BP4VA recorded on an electrochemical cell under a linear variation of the working silver electrode potential in conjunction with calculations of theoretical spectra with programs developed and implemented by us.The synthesis of 9,10-bis effect, which was discovered by Fleischmann 5 and uniform enhancement of the Raman spectrum, without modifying the relative intensities of the bands. For example, this is the case for the SERS spectra of pyridine registered on silver electrode at 0 and −0.25 V (λexc = 514.5 nm), where the relative intensities of the observed bands do not differ noticeably from those of the Raman spectra recorded in aqueous solution. In contrast, the chemical mechanism explains the selective intensification of specific bands of the Raman spectra.5The processes that are responsible for the signal enhancements in SERS are classified in two categories: (i) the electromagnetic (plasmonic/EM) or physical mechanism, which is always present in SERS, and (ii) the chemical mechanism (CM). Nowadays, it is widely accepted that localized surface plasmons (LSP) are responsible for EM mechanism. The EM mechanism explains the large (up to 10 magnitude orders)−–M+);12–23 (iii) surface-enhanced resonance Raman scattering (SERRS),24,25 when the laser wavelength matches the energy of an internal electronic transition of the adsorbed molecule; (iv) plasmon-like resonant mechanism (PL-SERS),26 a mechanism which was recently introduced by us and consists of a selective intensification of certain bands due to the coupling of internal resonant excitations of small metal clusters with specific normal modes of the adsorbate. It is important to note that, in essence, the SERS-CT contribution is part of the general SERRS process which involves the three mentioned different kinds of resonances (ii–iv); however, we introduce a distinction between them for the sake of clarity.The chemical contribution can be divided in several types: (i) effects due to the adsorption process of the molecule in its electronic ground state; (ii) charge-transfer mechanism (SERS-CT), which involves an internal resonant electronic transition of the complex formed by the adsorbed molecule (A) and the metal (M), in either of the two directions, A ↔ M, molecule to metal or metal to molecule  along with the calculated spectra of the four stable conformers optimized with the CAM-B3LYP/def2-TZVPP method. BP4VA is poorly soluble in water and it was not possible to record the aqueous solution Raman spectrum.27 The calculations of the minimum energy geometries predict that the isolated molecule has four stable conformers almost isoenergetic with different molecular symmetries (C2-symmetry geometry labeled as C2(a)  of anthracene (∼1640 cm−1), 8a of pyridine (∼1680 cm−1) and ν(CC) of vinylene (1739 cm−1), respectively, are selectively enhanced in accordance with the experimental behavior. Likewise, the calculations predict correctly the intensity ratios of such modes, being the 8a[1] mode of anthracene the most intense band of the triad. The calculated spectra also show that the 8a[2] mode (∼1477 cm−1) of anthracene should be selectively intensified as the densities of charge of the complexes becomes more negative as it is observed when the electrode potential in the experiments becomes more negative. At this point, it is important to note that the calculated SERS spectra resemble the resonance Raman spectra of the isolated species. The active bands are almost the same in both kind of spectra, excepting the 8a mode of pyridine, which is active in the SERS spectra but missing in the RR of the isolated species. A fact that is in accordance with the pyridyl moiety interacting with the metal through the nitrogen atom, as it is proposed in this work. In contrast, the calculated SERS differ noticeably with respect to the calculated and observed normal Raman spectra  of vinylene.3−–BP4VA) matches with the calculated intensities for the isolated species involving the neutral and the respective anion  states above and below the strongly allowed states which dominate the SERS spectra Table S2. Such CTnion top  and S6†,2–BP4VA–Ag2 -BP4VA or Ag2–Cs-BP4VA results. Therefore, we cannot distinguish between single or double union of the adsorbate to the metal. However, we can anticipate that the bidentate union is less probable because it requires a very specific structure of the local active sites of the metal.To finish, due to the adsorbate is a bidentate system, we have studied the SERS of BP4VA bonded to two symmetric silver clusters trough both pyridyl moieties: Ag2 Fig. S7. The speE)-2-(pyridin-4-yl)vinyl) anthracene  in a short time scale,43 SERS spectra have been recorded under 514.5 nm excitation by using a Renishaw Invia micro-Raman spectrometer. The microscope was equipped with a macro objective (f: 30 mm). Wire 2.0 from Renishaw has been used for spectral data acquisition and manipulation. The spectral resolution was set at 2 cm−1. The electrochemical instrument used to record the SERS spectra consists of a CH potentiostat (model 600E) and a three electrodes cell with (i) a platinum counter electrode, (ii) a Ag/AgCl/KCl sat. reference electrode to which all the electrode potentials were referred to, and (iii) a pure silver working electrode were used. The working electrode was electrochemically activated in order to produce the required SERS active nanostructures by maintaining the electrode potential at −0.5 V and then subjecting it to seven pulses at +0.6 V for 2 seconds. A 0.1 M aqueous solution of Na2SO4 has been employed as electrolyte in the activation procedure. Previously, the silver working electrode has been polished with 0.30 and 0.05 μm alumina (Büehler). SERS spectra of BP4VA/Na2SO4 with a concentration of 10−4 M/0.1 M were recorded. Pure water obtained from a Milli-Q instrument was employed in the solutions.BP4VA was synthesized by one of us (E. I.) as described in a recent article.The series of spectra, from 0.0 V up to −1.2 V, were recorded in triplicate being each spectrum recorded with the accumulation of 1 scan and a 10 s exposure time. The laser power at the sample was 6.0 mW. Spectra were not corrected for instrument response. There were no visible or spectroscopic signs of sample damage that were evident after data acquisition and also there were no significant fluctuations for a particular spectrum in all the sequence of spectra. The relative intensities of the bands for a particular spectrum at a specific electrode potential keep almost constant in the three series of spectra recorded, indicating a good reproducibility of the data.34–36 modified by a multi-state version44,45 which considers only Franck–Condon factors. It is assumed the small displacement approximation and the independent mode displaced harmonic oscillator (IMDHO) model. It is assumed as well that there is no mode rotation in the states under consideration.SERS and resonance Raman spectra have been calculated in accordance with the vibronic theory of Albrechtk,e, are obtained using the recurrence formulae of Manneback46 with the gradient approximation, fe is the force row vector of electronic state e, M is the 3N × 3N diagonal matrix of atomic masses, and Lk is the column eigenvector of the Hessian matrix for mode k-th.The integrals of vibrational overlapping, ΔK is a constant for a given experimental condition and a given irradiance of the incident radiation of wavenumber  PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.ngrt.i" format="PBM" resolution="300" x-size="9" y-size="11" xml:space="preserve">001100100010011000000000000000000000011000100001100110000100010000100100000101100000101000000110000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.454545pt" height="16.000000pt" viewBox="0 0 13.454545 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M160 840 l0 -40 -40 0 -40 0 0 -40 0 -40 40 0 40 0 0 40 0 40 80 0 80 0 0 -40 0 -40 80 0 80 0 0 40 0 40 40 0 40 0 0 40 0 40 -40 0 -40 0 0 -40 0 -40 -80 0 -80 0 0 40 0 40 -80 0 -80 0 0 -40z M80 520 l0 -40 40 0 40 0 0 -40 0 -40 40 0 40 0 0 -200 0 -200 80 0 80 0 0 40 0 40 40 0 40 0 0 40 0 40 40 0 40 0 0 80 0 80 40 0 40 0 0 80 0 80 -40 0 -40 0 0 40 0 40 -40 0 -40 0 0 -80 0 -80 40 0 40 0 0 -40 0 -40 -40 0 -40 0 0 -40 0 -40 -40 0 -40 0 0 -80 0 -80 -40 0 -40 0 0 200 0 200 -40 0 -40 0 0 40 0 40 -80 0 -80 0 0 -40z"/></g></svg>1; Ni0 is the Boltzmann population of the vibrational ground state; k is the wavenumber of k-th mode;  is given by μ0e; e, the energy difference between the excited electronic state e and the ground state; the wavenumber k of k mode and; the damping factor Γk,e which is related to the lifetime of the transition and is taken as 0.2k in this work. is the normalization constant given by To be specific, we have implemented a modified expression of the intensity equation given by Long, eqn (2)47 in conjunction with the def2-TZVPP basis sets for the first and second row elements and the corresponding electron core potential for the silver atoms.48,49 The metal–molecule complexes have been optimized in internal coordinates. The only constrain is that the geometries of the metallic clusters are kept frozen at the linear geometry calculated for the corresponding isolated systems. Each stationary point was characterized by its analytically computed harmonic frequencies. Mulliken's atomic charges have been used. The electronic structure calculations were carried out with the package of programs GAUSSIAN16.50 Adsorption energies were determined by using the standard expressions of statistical hermodynamics with programs implemented by us.51,52 Molecular structures, vibrational wavenumbers and molecular orbitals were analysed with the help of MacMolPlt,27 MOLDEN53 and GABEDIT54 programs.All of the quantum chemical calculations have been performed with the density functional theory by applying the hybrid exchange–correlation functional CAM-B3LYP,There are no conflicts to declare.RA-009-C9RA01269A-s001