Fe3O4/C composites were prepared by a simple one-pot hydrothermal method followed by a mild calcination procedure, using FeCl3∙6H2O as Fe3O4 precursor, and glucose as reducing agent and carbon source simultaneously. The Fe3O4/C composite modified glassy carbon electrode (Fe3O4/C/GCE) was prepared by drop-casting method. The microstructure and morphology of Fe3O4/C composite was characterized by powder X-ray diffraction (XRD) and scanning electron microscopy (SEM), respectively. Due to large specific surface area and synergistic effect from Fe3O4 nanoparticles and carbon coating, Fe3O4/C composite showed excellent electrocatalytic activity toward the oxidation of Trp. As a result, the proposed Fe3O4/C/GCE displayed superior analytical performances toward Trp determination, with two wide detection ranges (1.0–80 μM and 80–800 μM) and a low detection limit . Moreover, successful detection of Trp in human serum samples further validate the practicability of the proposed sensor.In this study, we reported facile synthesis of Fe Tryptophan (Trp), one of eight essential amino acids in human body, plays indispensable roles in maintaining human growth, metabolism, and positive nitrogen balance . Trp is At present, various analytical techniques have been developed for the identification and determination of Trp, including but not limited to high-performance liquid chromatography , capilla2 ), potassium ferrocyanide (K4[Fe(CN)6]), and ethyl alcohol were supplied by Sinopharm Chemical Reagent Co., Ltd. . All these chemicals were used as received without further treatment. A series of different concentrations of Trp standard solutions was prepared by appropriately diluting 1 mM stock solution of Trp with 0.1 M phosphate buffer saline solution . Ultrapure water (18.2 MΩ) was used throughout the experiments.L-tryptophan (Trp) was purchased from Aladdin Bio-Chem Technology Co., LTD . Human serum samples were taken from People’s Hospital of Zhuzhou. Ferric trichloride hexahydrate . Then, they were alternately washed by ethyl alcohol and ultrapure water under ultrasonication for 2~3 times (each for 1 min), respectively. Five microliters of Fe3O4/C dispersion (1 mg/mL) was drop-casted onto the surface of polished GCE, then dried under an infrared lamp to obtain Fe3O4/C composites modified GCE (Fe3O4/C/GCE). For comparison, pure Fe3O4 NPs modified GCE (Fe3O4/GCE) was also prepared by the similar method. At first, the glassy carbon electrodes (GCEs) were polished by α-AlScanning electron microscopy (SEM) images were collected from a Hitachi S4800  operating at 5 kV. Powder X-ray diffraction (XRD) patterns were recorded on an X-ray diffractometer  operating at 40 kV and 40 mA with Cu Kα radiation (λ = 0.1542 nm).3O4/C/GCE were investigated in a freshly prepared 0.1 M PBS containing 10 μM Trp by cyclic voltammetry (CV). The quantitative analysis of Trp were performed by second derivative linear scan voltammetry (SDLSV). CV and SDLSV were recorded on an electrochemical workstation  and a Polarographic Analyzer , respectively. Both CVs and SDLSVs were measured using a conventional three-electrode assemble, in which bare or modified GCEs worked as working electrode, while platinum wire electrode and saturated calomel electrode (SCE) acted as counter electrode and reference electrode, respectively. Before all the electrochemical measurements for Trp, a suitable accumulation was performed under stirring at 500 rpm to increase the electrochemical responses. After a 5 s rest, both the CVs and SDLSVs were measured at a scanning rate of 0.1 V/s. The potential was scanned from −0.2 to 1.2 V for both CV and SDLSV.The electrochemical behaviors of as-prepared FeUnder the optimal voltammetric conditions, Trp in human serum was detected using SDLSV technique. Typically, 1 mL human serum sample was diluted to 10 mL with 0.1 M PBS (pH 6.5). Standard addition method was further used to validate the reliability of the proposed sensor. 3O4 NPs and Fe3O4/C composite were characterized by power X-ray diffraction (XRD) technique. As presented in 3O4 NPs and Fe3O4/C composite, respectively. The emerged various sharp peaks indicating that these as-prepared samples show good crystallization performance. Moreover, the diffraction peaks located at 30.063°, 35.451°, 43.037°, 53.546°, 57.166°, and 62.726° are corresponding to crystal facets of (220), (311), (400), (422), (511), and (440), respectively. This result is in good accordance with standard PDF card of magnetite . The corresponding peaks of magnetite in the Fe3O4/C sample are also apparent, indicating that a good crystallization is observed. Moreover, the crystallization is not influenced by carbon coating. Besides, a wide peak located at 20°~30° is observed, which could be ascribed to diffraction peak of amorphous carbon layer. These results confirm that the Fe3O4/C nanocomposite was synthesized successfully.The microstructures of pure Fe3O4 NPs and Fe3O4/C nanocomposites were further characterized by scanning electron microscopy (SEM). As presented in 3O4 NPs are observed with excellent dispersity, and the size of these Fe3O4 NPs is roughly estimated as 200~300 nm. However, in the Fe3O4/C composite sample, the particle size is larger than that of pure Fe3O4 NPs 6  are observed at the bare GCE, with peak-peak separation (∆Ep) of 0.153 V. At the Fe3O4/GCE, the redox peak currents  are inferior to those of bare GCE, probably because of the poor electrical conductivity of Fe3O4 NPs. Moreover, the peak-peak separation (∆Ep) decrease to 0.145 V, suggesting Fe3O4 NPs effectively reduced the active polarization. Among these three electrodes, Fe3O4/C/GCE shows the largest response peak currents  with lowest peak-peak potential separation (∆Ep = 0.079 V), due to the synergistic effect from high electrocatalytic activity of Fe3O4 NPs and excellent electrical conductivity of carbon shells. The cathodic peak currents (ipc) of bare GCE, Fe3O4/GCE, and Fe3O4/C/GCE are 14.71 μA, 12.61 μA, and 25.29 μA, respectively. Referring to the Randles-Sevcik equation [ipc represents the cathodic peak currents of K3[Fe(CN)6], n represents the transferred electron number during the redox reaction of K3[Fe(CN)6], D is the diffusion coefficient of K3[Fe(CN)6] (7.6 × 10−6 cm2 s−1  (mol cm−3). The electrochemical active areas of bare GCE, Fe3O4/GCE and Fe3O4/C/GCE are calculated as 0.01255 cm2, 0.01075 cm2, and 0.02209 cm2, respectively. The electrochemical active area of Fe3O4/C/GCE is about two-fold that of bare GCE, indicting that the Fe3O4/C composite modification could enhance the effective electroactive surface area, which can facilate the adsorption of target analytes on the electrode surface and offer more catalytic active sites, and thus accelerate the redox process of target analytes. The CVs of bare GCE, Fe3Fe(CN)6 . A pair equation ,47,48:ip cm2 s−1 ), v is t3O4/GCE, and Fe3O4/C/GCE electrodes were investigated by CV (ipa = 4.603 μA) is observed at 0.929 V. After the modification of GCE with Fe3O4 NPs, the ipa of Trp increases to 7.414 μA, suggesting Fe3O4 NPs have high electrocatalytic activity toward Trp oxidation. However, the anodic peak shifts positively to 0.963 V, due to the poor electrical conductivity of Fe3O4 NPs. When decorated with Fe3O4/C nanocomposites, a sharp anodic peak appears with highest response peak current (13.13 μA) and lowest working potential (0.750 V). The ipa of 10 μM Trp at Fe3O4/C/GCE is about two times higher than that at bare GCE. The enhanced anodic peak current and decreased anodic peak potential is mainly due to the synergistic effect of Fe3O4 NPs and carbon coating. Specifically, Fe3O4 NPs offer favorable catalytic active sites for the Trp oxidation; The carbon coating improves the electrical conductivity and decreases the charge transfer resistance (Rct). Besides, the large electrochemical active area of Fe3O4/C composite also contributed to the enhancement on the ipa of Trp. The SDLSV responses of 10 μM Trp at various electrodes are shown in ipa = 9.852 μA) occurs at 0.852 V. When modified with Fe3O4 NPs, the anodic peak shifts to 0.901 V and the anodic peak current increases to 16.42 μA. After the modification of GCE with Fe3O4/C composites, a well-shaped anodic peak was observed with largest anodic peak current of 46.55 μA and lowest anodic peak potential of 0.746. The SDLSV results are highly consistent with the CV results. Obviously, SDLSV is more sensitive than CV for Trp detection. So, SDLSV was performed for the detection of Trp.The electrochemical responses of 10 μM Trp on bare GCE, Feed by CV a. Clearlipa increases with pH varying from 5.5 to 6.5, then gradually declines with pH further increasing. So, pH 6.5 was selected for the subsequent measurements. Moreover, the anodic peak potential (Epa) is highly linear to solution pH, and the corresponding linear regression equation is Epa(V) = −0.061pH + 1.234 with correlation coefficient (R2) of 0.994. The slope (−61mV/pH) is very close to theoretical value from Nernst equation (−59 mV/pH), suggesting the equal number of protons (H+) and electrons (e−) are involved in the electrochemical oxidation of Trp [In order to investigate the influence of solution pH, the CVs of 10 μM Trp were measured in various pH PBS a. As plon of Trp . −1) in 0.1 M PBS (pH 6.5), aiming to reveal the electrooxidation mechanism of Trp on Fe3O4/C/GCE. As can be seen from ipa of Trp increases with the speeding of scanning rates, but the background currents also increase with the increase of the scanning rates. More importantly, a good linear relationship between anodic peak currents (ipa) and scanning rates (lnv) is found in pai (μA) = 6.479v (V/s) + 2.582 (R2 = 0.982). It demonstrates the electrooxidation of Trp on Fe3O4/C/GCE is an adsorption-controlled process [v), and the linear equation can be expressed as Epa(V) = 0.03593lnv + 0.9500 (R2 = 0.990). As for an adsorption-controlled and totally irreversible electrode process, it should follow the Lavrion’s theory as follows [Ep denotes the peak potential, E0 denotes the formal potential (V), R denotes the gas constant (8.314 J K−1 mol−1), T denotes the temperature (298.15 K), α denotes the charge transfer coefficient, n represents the electron transferred number, F is the Faraday constant , k0 represents standard heterogeneous transfer rate (s−1), and v is the scanning rate (V/s), respectively. Referring to Laviron’s equation, the slope of the linear equation is equivalent to RT/αnF  and electrons (e−) participate in the electrochemical oxidation of Trp. So, the electrooxidation of Trp at Fe3O4/C/GCE is a two-electrons and two-protons process . The electrochemical mechanism of Trp oxidation is described in The CV curves of 10 μM Trp were recorded at different scanning rates 0.03~0.30 V s V s−1 in process ,33. As s follows :(2)Ep=E0 process ). Hence,3O4/C/GCE is an adsorption-controlled process. The Trp was accumulated on the surface of Fe3O4/C/GCE at various potentials for 240 s, then their ipa of were measured by SDLSV. As presented in ipa is achieved at 0.2 V. Hence, 0.2 V was recommended as the optimal accumulation potential. Moreover, the influence of accumulation time on the ipa were also investigated at fixed accumulation potential of 0.2 V  = 0.2120c (μM) + 0.4406 (R2 = 0.986) and ipa (μA) = 0.03746c (μM) + 14.37 (R2 = 0.979), respectively. The slopes of two linear regions are very different, because the contributions of adsorptive and diffusion current components are changed in 3~4 orders within the concentration range. The LOD was estimated according to the equation: LOD = 3σ/s, where σ is the standard deviation of the blank solution and s is the slope of the calibration plots. The LOD (S/N = 3) is estimated as 0.26 μM. The superb sensing performance is mainly due to synergistic effect from high catalytic activity of Fe3O4 NPs and excellent conductive carbon coating. A comparison of analytical performances between our proposed sensor and the existing electrochemical sensors is summarized in 3O4/C/GCE has outstanding advantages including facile preparation and low costs, which is very important for the practical applications. SDLSV curves of various concentrations of Trp are shown in .0–80 μM c and 80–.0–80 μM d. The co6 R2 = 0.86 and ippa of 10 μM Trp (n = 10), demonstrating good repeatability. The reproducibility was also checked by paralleled measurements of 10 μM Trp, using five Fe3O4/C/GCEs fabricated by the same procedures. The RSD  is accepted, suggesting highly reproducible for electrode preparation. Finally, the stability was assessed by monitoring the anodic peak currents of 10 μM Trp at a same Fe3O4/C/GCE during a week. When not in use, Fe3O4/C/GCE was immersed in Trp solution and stored at a 4 °C refrigerator. After a week, the anodic peak current retains 90.4% of the original response. The repeatability and reproducibility assays were also carried out to verify the practicability. The repeatability was examined by ten successive measurements of the i0 μM Trp . The reln = 3). Moreover, the good recoveries (105–107%) shows the proposed Fe3O4/C/GCE has great application prospects in the detection of Trp from various real samples.SDLSV was adopted for quantitative analysis of Trp due to its superior sensitivity and high resolution ,31,33. T3O4/C composite and its application for cost-effective and sensitive detection of tryptophan (Trp) in human serum samples. The Fe3O4/C/GCE was prepared by drop-casting method. Owing to the large electrochemical active area and synergistic effect from Fe3O4 NPs and carbon coating, the response anodic peak current enhanced greatly. As a result, the proposed Fe3O4/C/GCE showed two wide linear detection ranges (1.0–80 μM and 80–800 μM) and a low LOD of 0.26 μM. Finally, the proposed Fe3O4/C/GCE successfully realized the detection of Trp in human serum samples with satisfactory recoveries (105–107%). Together with facile preparation and low cost, Fe3O4/C/GCE has shown tremendous application prospects on the detection of Trp from various actual samples.In this study, we reported facile one-pot synthesis of Fe


E123IR makes intramolecular interactions restraining Atg3′s catalytic loop, while E1 and E3 enzymes directly remove this brace to conformationally activate Atg3 and elicit Atg8 lipidation in vitro and in vivo. We propose that Atg3′s E123IR protects the E2~UBL thioester bond from wayward reactivity toward errant nucleophiles, while Atg8 lipidation cascade enzymes induce E2 active site remodeling through an unprecedented mechanism to drive autophagy.Autophagy depends on the E2 enzyme, Atg3, functioning in a conserved E1-E2-E3 trienzyme cascade that catalyzes lipidation of Atg8-family ubiquitin-like proteins (UBLs). Molecular mechanisms underlying Atg8 lipidation remain poorly understood despite association of Atg3, the E1 Atg7, and the composite E3 Atg12–Atg5-Atg16 with pathologies including cancers, infections and neurodegeneration. Here, studying yeast enzymes, we report that an Atg3 element we term E123IR  is an allosteric switch. NMR, biochemical, crystallographic and genetic data collectively indicate that in the absence of the enzymatic cascade, the Atg3 Autophagy mediated by the conjugation pathway for ubiquitin-like proteins plays a key role in controlling homeostasis in eukaryotic cells. Here the authors provide a molecular basis for allosteric activation of the E2 ligase Atg3, uncovering the mechanism underlying Atg8 lipidation and a novel mechanism regulating E1-E2-E3-mediated ubiquitin-like protein conjugation. Sectors of the cytosol are first sequestered within a double-membrane-bound autophagosome, and then degraded upon autophagosome fusion with the lysosome in higher eukaryotes or vacuole in yeast2. Cargoes directed to autophagosomes for degradation, include protein assemblies, organelles, misfolded macromolecules, debris, and pathogenic bacteria5. As such, autophagy plays important roles in cellular metabolism in many physiological processes, while dysregulation of autophagy is associated with metabolic, neurologic, and oncologic disorders12.Autophagy is a major catabolic pathway by which eukaryotic cells degrade and recycle diverse cellular materials16. Ultimately, lipidated Atg8-family members serve as bridges between membranes and the cytosol, where their ubiquitin-like fold domains recruit Atg8-interacting motif (AIM) or LC3-interaction region (LIR) sequences in a staggering array of partner proteins involved in various aspects of autophagy20. These, include regulators and effectors of autophagosome biogenesis that promote membrane tethering and growth of the autophagosome, as well as adaptor proteins that in turn recruit cargoes for degradation21. Accordingly, Atg8 and its conjugation system enzymes are essential for autophagy in yeasts, and autophagy is severely impaired in their absence in mammalian cells22. Lipidated Atg8-family members have also been implicated in protein recruitment to membranes in processes that are unrelated to autophagy in mammalian cells23. Therefore, given these important roles of Atg8 and its conjugation system enzymes, it is important to understand molecular mechanisms regulating Atg8 lipidation.Many facets of autophagy rely on the autophagy-specific ubiquitin-like protein (UBL) Atg8 in yeast  and the E1–E2–E3 enzymatic cascade that links Atg8′s C-terminus to the primary amino head group of phosphatidylethanolamine (PE) lipid molecules24. Atg3 receives Atg8 from the E1 Atg7, to form a covalent, reactive Atg3~Atg8 intermediate linked by a thioester bond (referred to by ~) between the C-terminus of Atg8 and the catalytic Cys of Atg3. Atg8 is then transferred from the Atg3 catalytic Cys to PE in a reaction catalyzed by a composite E3 enzyme25 containing another autophagy UBL, Atg12, conjugated to Atg526. In vitro, the yeast Atg12–Atg5 conjugate is sufficient to serve as an E3 enzyme activating lipidation of Atg826. Atg16 binding to lipids substantially enhances E3 activity27 and is required in vivo28 to localize Atg12–Atg5 to the preautophagosomal structure that precedes autophagosome formation30, through many mechanisms33.The central enzyme in the Atg8 lipidation cascade is the E2, Atg3S. cerevisiae revealed a catalytic core domain (Atg3cat) largely resembling E2s for canonical UBLs such as Ub, NEDD8 and SUMO, including a long central helix and two catalytic segments34 ; an intrinsically disordered flexible region ; a handle region consisting of a helix and a disordered region  that contains an Atg8-interacting motif that is dispensable for the lipidation reaction but plays important roles in vivo; and a flexible extreme C-terminal extension . While prior structural studies showed how Atg7 engages Atg3cat and the Atg3FR for Atg8 transfer to Atg343, less is understood about subsequent roles of the autophagy E3. A crystal structure showed how the human E3 enzyme recruits a peptide-like region from Atg3′s FR, but this FR motif is not conserved across Atg3 sequences from many organisms, including yeasts44. Furthermore, the structure lacked Atg3′s catalytic domain, for which prior studies of cysteine accessibility in the yeast enzyme suggested that the autophagy E3 triggers a structural rearrangement that affects the orientation of the catalytic Cys234 relative to Thr21335. The molecular underpinnings for E3-dependent conformational activation remains elusive.The prior structure of Atg3 from s34 Fig. . One segHere, we use a hybrid structural approach integrating information from biochemistry, nuclear magnetic resonance (NMR), crystallography, and yeast genetics to identify a molecular basis for Atg3 allosteric activation. Overall, the data suggest that a distinct peptide-like element acts like a brace mediating intramolecular interactions with the Atg3 catalytic domain to restrict the conformation of the active site and prevent reactivity toward errant nucleophiles, while the E3 elicits an unprecedented E2 activation mechanism by remotely binding to this brace to couple other E3 interactions with allosteric activation of active site mediating Atg8 lipidation.47. Many E3s, for example RING and related E3s for UB, NEDD8 and SUMO, and SIM-containing SUMO E3s, allosterically promote noncovalent interactions between the E2 and UB (or UBL) moieties within the covalent intermediate. This is often monitored by an E3 increasing the susceptibility of an E2–UB or E2–UBL intermediate to nucleophilic attack by a nonspecific amine nucleophile48. In contrast, HECT and RBR E3s use a different mechanism that depends on positioning the E2-bound UB for transfer to the E3 catalytic Cys. Although the autophagy E3 lacks homology to UB, NEDD8, and SUMO ligases, we asked whether there are common mechanistic features by testing biochemical activities of the relatively simplistic Atg8 pathway from S. cerevisiae. Potential E3-mediated stimulation of intrinsic activity of the Atg3~Atg8 intermediate was examined by adapting the pulse-chase assay format used to study canonical UB enzymes , an unlabeled version of the Atg12–Atg5 conjugate was added, and  TROSY spectra were examined for chemical shift perturbation and/or line-broadening as an indicator of binding  anchors the complex, thereby increasing the local concentration for distal, transient interactions between Atg7′s and Atg3′s catalytic domains for Atg8 transfer between them43  previously shown to impair Atg3 binding   that are important for the lipidation reaction  Fig. . Howeverion Fig. , right34 HR Fig. ; the rem HR Fig. , left. M FR Fig. .Fig. 5Co43. Although interpreted as stemming from indirect Atg7 interactions with the backside of Atg3′s catalytic domain43, and/or from the high pH of the crystallization conditions35, reinterpretation of the crystal structures with the knowledge that the Atg3E123IR element binds Atg3′s catalytic domain in solution leads to a prediction: interactions with Atg3E123IR would allosterically restrict Atg3′s catalytic domain. However, displacement of the Atg3E123IR element, either by deleting the FR domain . Upon adding this to the 15N-labeled Atg3FR, we observed the similar chemical shift perturbations in  TROSY spectra of 15N-labeled Atg3FR  genes have been deleted (MKO)nts Fig. , which iTo identify the portions of Atg8 and the Atg3 catalytic domain contributing to the activated state, we performed mutagenesis. E3-dependent discharge was monitored for 28 mutant versions of Atg8 and 27 mutant versions of Atg3, each having one to four Ala replacements for surface residues Fig. . The Atg15N, 1H] TROSY spectra for 15N-labled Atg8 harboring a C-terminal Cys alone vs. in a disulfide-bonded complex linked to the active site of unlabeled Atg3cat—i.e., in a stable proxy for the Atg3cat–Atg8 intermediate  Atg7′s N-terminal domain binds to Atg3′s E123IR element. This both mediates E1–E2 interactions and conformationally activates Atg3′s Cys to receive Atg8 from Atg7. (3) Following formation of the Atg3~Atg8 intermediate, Atg3′s E123IR element could re-engage the Atg3 catalytic domain to prevent Atg8 mis-ligation to a nonspecific nucleophile. We speculate that this may also prevent the extreme C-terminal residues of Atg3 from prematurely adopting a conformation that contributes to the activate conformation of the Atg3~Atg8 intermediate. (4) Binding with relatively higher affinity, the Atg12–Atg5 portion of the autophagy E3 subsequently dislodges Atg3′s E123IR, which enables structural remodeling of the active site loop from a sequestered position to an exposed conformation where the catalytic Cys is juxtaposed with side chains that are crucial for Atg8 lipidation  Prior to encountering the Atg7–Atg8 intermediate, the Atg3ion Fig. . Apparen37. The active site of Atg3~Atg8 may require protection to prevent inopportune discharge to PE, and this is best ensured by coming from the intermediate itself. Indeed, when conformationally activated by E3-mimicking mutations, Atg3~Atg8 cannot only catalyze the lipidation reaction and in vivo, but also robustly discharges Atg8 to nonspecific nucleophiles even in the absence of E3. Thus, our data provide a mechanism ensuring that Atg8 discharge is coupled to binding to E3, which in turn establishes targeting specificity through several features, including binding to cargo receptors56, and to membranes31. The Atg16 moiety in the autophagy E3 could subsequently engage the lipidated Atg8 to serve as a scaffold during autophagosome biogenesis32.Why might the autophagy UBL pathway involve such elaborate allosteric control? It seems likely that key features of the ultimate target, and intrinsic capabilities of the Atg3~Atg8 intermediate, impose distinctive requirements for regulation. First, the substrate PE is among the most abundant core glycerophospholipids. Found in several organelles, PE’s widespread accessibility differs from canonical UB and UBL substrates, which typically only are licensed for modification after a regulatory event such as a post-translational modification directs E3 binding. Second, through its N-terminal membrane-curvature sensing helix, Atg3 itself can bind the substrate PE47. It is now well-recognized that many E3 catalytic domains stabilize a closed conformation that renders E2–UB and E2–UBL intermediates susceptible to nucleophilic attack, while other E3 domains mediate substrate targeting. Many E2s are further activated allosterically, through their backside beta-sheets binding to noncatalytic E3 domains. These effects have largely been characterized for indirectly increasing binding to E3 RING domains. However, some E3 or E3-associated domains also trigger conformational changes at E2 active sites. For example, the membrane E3-associated Cue1p binds to Ubc7p’s backside, and indirectly modulates the active site, tens of angstroms away57. Irrespective of whether E2 cysteines require protection from errant nucleophilic attack, or coordination of catalytic activation with other E3 functions, it seems likely that allosteric modulation of E2 active sites serves as a failsafe ensuring UB and UBL discharge at appropriate targets. The autophagy E2 Atg3 now surfaces as an ultimate case, with its brace self-imposed until directly lifted by the E3 to drive autophagy.Allosteric E2 activation is emerging as a common property of E3 ligasesScAtg3 and HsATG3 protein sequence alignment are done by CLUSTALW online.58. For Atg7 proteins, full-length Atg7 or a fragment containing residues 1–289 (NTD) was inserted into the pGEX-4T-1 vector following the Gibson Assembly protocol59, and transformed into BL21 competent Escherichia coli; protein expression and purification protocols for Atg7 WT and mutants resemble those for Atg3 and Atg8. To obtain purified E3 complex, N-term 6X His-tagged Atg12 100–186 and untagged Atg5 1–284 were inserted into the two multiple cloning sites (MSCs) of pRSF-Duet-1, respectively, whereas Atg7 1–630 and Atg10 were inserted into the MSCs of pET-Duet-1. The two plasmids are co-transformed into BL21 competent E. coli to produce His-Atg12–Atg5. Bacteria culture were grown at 37 °C to reach O.D. 1.5, followed by 0.6 mM IPTG induction at 23 °C overnight. Cells are harvested in 1× phosphate-buffered saline, 400 mM NaCl and lysed by sonication. Ni-NTA resin (Qiagen) was incubated with lysate for 1 h then washed 3 times with the same buffer. His-Atg12–Atg5 were eluted by buffer with 200 mM imidazole and immediately purified by cation exchange chromatography on an S column . Purified his-Atg12–Atg5 were combined with Atg16 1–46 in a 1:2 molar ratio and further purified by size-exclusion chromatography on a Superdex-200 column  to harvest peak factions containing the final product of His-Atg12–Atg5-Atg16.Detailed methods for cloning, expressing and purifying yeast proteins Atg8, Atg3 (and their mutants or truncated versions), and Atg16 (residues 1–46) were described previously∆NFR protein sample was manually loaded onto a glass hanging-drop cover and overlaid with 1 µL of precipitant solutions. Crystal was harvested from 100 mM HEPES pH 7.2, 150 mM l-malic acid, 9% PEG3350. Crystals were allowed to grow at 4 °C for 5–7 days to reach their maximum sizes. All crystals were harvested by loops with 25% glycerol in addition to growth solution, and stored in liquid nitrogen for 2 days until data collection.Totally, 1 µL purified Atg3∆NFR was collected at Advanced Photon Source (APS) Ser-CAT beamline 22-BM. The Dataset was created from single crystals, and was processed and scaled in HKL2000. Phase was solved by molecular replacement. Search model for Atg3∆NFR was modified from Atg3FL (PDB 2DYT) with Atg3E123IR manually removed. Model was built in Coot60, and refinements were primarily performed in Refmac61 and finalized in Phenix62.Data for Atg362 with simulated annealing running from 5000 to 300 K in 50 steps. Finally, the Fo–Fc map was shown at 3σ level along with the deleted residues in the model.In order to avoid the model bias, we generated a simulated annealing omit map for the catalytic cysteine region. First, residue 231–237 in the final-refined model was deleted; then, the model with deletion was subjected to 3 rounds of refinement in PhenixAll the cysteines except C234 within Atg3 were mutated to alanines. 800 µM Atg8 K26P C22V G116C was reduced by 20 mM DTT, desalted into buffer 1 , and immediately mixed with 1:1 v/v buffer 2 . DPS stock was pre-dissolved in a minimal amount of DMSO), incubated at room temperature (RT) for 20 min, and desalted again into buffer 1. Meanwhile, 500 µM Atg3 was reduced by 20 mM DTT and desalted into buffer 1, immediately mixed with DPS-Atg8 at a 1:2 molar ratio, and incubated at RT for 1 h. Crosslinking product was purified by anion exchange chromatography on Q column  and size-exclusion chromatography on a Superdex-200 column .2O on either a Bruker Avance 600-MHz or 700-MHz or 800-MHz spectrometer equipped with a 5-mm triple-resonance cryoprobe and a single-axis pulse field gradient. For resonance assignments of backbone atoms, 3D triple-resonance experiments were measure either with 0.4 mM 13C, 15N, 2D-labeled or 13C, 15N-labeled Atg3cat, 0.75 mM 13C, 15N-labeled Atg3FR 86–159, and 0.4 mM 13C, 15N-labeled Atg8 K26P, C33V, G116C. The backbone assignment of Atg3cat was carried out using 3D HNCACB, HNcoCACB, HNCO, HNcaCO along with 15N-resolved  NOESY spectra. The backbone of Atg3FR 86–159 fragment peptide was assigned using 3D HNCACB, CACBcoNH, HNCO, HNcaCO along with HNcocaNH spectra. The backbone assignment of Atg8 K26P, C33V, G116C was carried out using 3D HNCACB, HNcoCACB, HNCO, HNCA and HNcoCA along with 15N-resolved  NOESY spectra. Spectra were processed with Topspin 3.5 or nmrPipe and analyzed using CARA. All chemical shift perturbation analyses were done using  TROSY spectra, and the perturbations were calculated using the formula CSP (ppm) = ((δH)2 + 0.2*(δN)2)0.5. Titration experiments were done using 0.1 mM 15N-labeled Atg3FR 86–159  titrated with 0–150 µM Atg12–Atg5 /(x + Kd), where x is the concentration and Kd is the dissociation constant and y0 is a constant.All the experiments were conducted at 25 °C with protein samples dissolved in 20 mM MES pH 6.5, 100 mM NaCl, and 10% v/v Dtg5 Fig. , 0–500  was dissolved in chloroform, solvent evaporated using a nitrogen stream, and re-dissolved in 250 µL 20 mM HEPES pH 7.0, 150 mM NaCl buffer. Product was passed through 100 nm nuclepore membrane (Whatman) installed in a mini extruder (Avanti) 15 times to obtain 10 mg/mL homogenized liposomes in solution.2.5 mg of 2 were mixed in 25 µL 20 mM HEPES pH 7.0, 150 mM NaCl buffer. After 1 h, pulse reactions were quenched by adding Apyrase. In chase step, 5 µL of each pulse product was incubated with 5 µL chase solution containing 5 µM Atg12–Atg5-Atg16 and a course of NH2OH at RT for 2.5 min, or with liposome at a final concentration of 2 mg/mL added in a timely manner. Samples were analyzed by 4–12% Bis–Tris Protein Gels (Thermo Fisher Scientific) and stained with Coomassie Brilliant Blue. All raw data are available in the source data file.Shortly before the starting the experiment, Atg3 and mutants were reduced with 20 mM DTT and desalted by Zeba columns (Thermo Fisher Scientific) into reaction buffer. In the pulse step, 5 µM Atg7, 20 µM Atg3, 40 µM Atg8, 1 µM ATP, and 1 µM MgCl2 and 1 mg/mL liposome, and were monitored in timely manner. Totally, 6 µL of each reaction was mixed with 6 µL 2× protein loading buffer. Samples were analyzed using a by 15% urea gel at 4 °C and stained with Coomassie Brilliant Blue. All raw data are available in the source data file.Atg3 and mutants were reduced and desalted as described in the pulse-chase assay. Reactions were performed at 30 °C in 25 µL volumes of 20 mM HEPES pH 7.0, 150 mM buffer, with 1 µM Atg7, 2 µM Atg3, 5 µM Atg8, 5 µM Atg12–Atg5-Atg16, 1 mM ATP, 1 mM MgCl55 cells were transformed with pRS416 vectors harboring HA-tagged Atg3 (WT or mutants); the YCY131 cells were co-transformed with pRS414–Atg7–Atg10–Atg8∆R. For nutrient-rich conditions, yeast cells were grown in synthetic minimal  medium. For nitrogen starvation, cells were first cultured in appropriate SMD medium to mid-log phase; they were then shifted to the SD-N medium . The XLY161 cells were cultured in SMD-Ura to mid-log phase before they were shifted to SD-N medium for 2 h. The YCY131 cells were cultured in SMD-Ura-Trp to mid-log phase before they were shifted to SD-N medium for 4 h. All raw data are available in the source data file.Yeast strains used in this study are listed in the table below. The XLY161 and the YCY131One OD of each of the yeast cultures was harvested and lysed in 50 μl MURB  by vortexing them with glass beads at 4 °C for 5 min. The samples were then heated at 70 °C for 10 min before they were loaded onto and run in SDS-PAGE gels, followed by transferring onto polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk, probed with primary antibodies , and then probed with horseradish peroxidase (HRP)-coupled secondary antibodies . Atg8 antiserum was generated by injecting purified yeast Atg8 peptides into rabbits; Dpm1 antibody is purchased from Molecular Probes ; antibody to the HA epitope is from Sigma ; Pgk1 antibody is a generous gift from Dr. Jeremy Thorner, University of California, Berkeley. Goat anti-rabbit HRP sencondary antibody is from Fisher , and rabbit anti-mouse HRP sencondary antibody is from Jackson ImmunoResearch . Chemiluminescence images were obtained by developing films in a dark room. The standard-sized protein bands from the protein ladder lanes on the blots were correspondingly hand-drawn using a pen onto the developed films. The hand-drawn protein ladders were not shown in the figures, but they can be found in the source data file.Further information on research design is available in the Supplementary InformationPeer ReviewReporting SummarySource Data


Saccharomyces cerevisiae, phosphorylation of H4 threonine 80 (H4T80ph), and show that it triggers checkpoint inactivation. H4T80ph is critical for cell survival to DNA damage, and its absence causes impaired DDC recovery and persistent cell cycle arrest. We show that, in response to genotoxic stress, p21-activated kinase Cla4 phosphorylates H4T80 to recruit Rtt107 to sites of DNA damage. Rtt107 displaces the checkpoint adaptor Rad9, thereby interrupting the checkpoint-signaling cascade. Collectively, our results indicate that H4T80ph regulates DDC recovery.In response to genotoxic stress, cells activate a signaling cascade known as the DNA damage checkpoint (DDC) that leads to a temporary cell cycle arrest and activation of DNA repair mechanisms. Because persistent DDC activation compromises cell viability, this process must be tightly regulated. However, despite its importance, the mechanisms regulating DDC recovery are not completely understood. Here, we identify a DNA-damage-regulated histone modification in   •Histone H4T80 phosphorylation regulates DNA damage checkpoint recovery•In response to DNA damage, PAK family kinase Cla4 phosphorylates H4T80•H4T80 phosphorylation promotes timely recruitment of the scaffold protein Rtt107•H4T80ph-bound Rtt107 counteracts Rad9 binding to H3K79me Termination of the checkpoint-signaling cascade is essential for resumption of normal cell growth after DNA damage. Millan-Zambrano et al. show that phosphorylation of histone H4T80 regulates the transition between DNA damage checkpoint activation and recovery by promoting timely recruitment of Rtt107 protein. Genome integrity is continuously threatened by DNA damage arising from both exogenous and endogenous sources. However, the DNA damage response (DDR) pathway identifies and repairs damaged DNA to ensure that the genetic information is faithfully maintained. The eukaryotic genome is compacted into chromatin, whose fundamental repeating unit is the nucleosome. Nucleosomes consist of 147 base pairs of DNA tightly wrapped around a core histone octamer, which is composed of two copies of histones H2A, H2B, H3, and H4 . ImportaHistone post-translational modifications occur both in the tails and the core domains. One of the first-studied core modifications was methylation of histone H3 lysine 79 (H3K79me), which plays an important role in the DDR . Since tSaccharomyces cerevisiae, Mec1 kinase orchestrates the DDC signaling process. Mec1 is first recruited to single-stranded DNA (ssDNA) through an interaction with replication protein A , which results in a temporary cell cycle arrest and activation of DNA repair pathways . The molrotein A  and thenrotein A . Two dirrotein A , which irotein A . Rad9 plrotein A . Once furotein A .Whereas the molecular mechanisms regulating DDC activation are well understood, comparatively less is known about how DDC recovery is initiated and controlled. Even though different factors have been shown to be involved in the process , the molSaccharomyces cerevisiae and investigated phenotypes of resulting strains. We observed that, in contrast to other mutations analyzed, mutation of either arginine 78 (H4R78A) or threonine 80 (H4T80A) results in severe hypersensitivity to the DNA-damaging agents camptothecin (CPT) and methyl methanesulfonate (MMS)  A.Figure We initially focused on the possible role of H4R78 in the DDR. H4R78 forms hydrogen bonds with histone H4 aspartate 85 (H4D85) A, but diAlthough H4T80 could exert its functions in various ways, we were intrigued by the possibility that it might be phosphorylated in a manner requiring a basic amino acid residue at position 78. In this regard, certain basophilic protein kinases are known to target either threonine or serine preceded by basic residues . In agreDifferent large-scale mass spectrometry studies identified histone H4T80ph in higher eukaryotes . HoweverBecause the H4T80A mutant is sensitive to DNA-damaging agents, we asked whether H4T80ph levels might be regulated in response to DNA damage. Importantly, immunoblot studies confirmed that H4T80ph levels increase upon CPT treatment D. To invWe next sought to identify the kinase(s) responsible for H4T80ph. Although yeast basophilic kinases commonly show a strong selectivity for arginine residues three amino acid residues upstream of the phosphorylation site (P-3), only four of them  are selective for arginine at the P-2 position . As the CLA4, but not SKM1 or STE20, results in DNA damage hypersensitivity  and a kinase dead version  in mammalian cells , which is completely dependent on Rad52 . If the DNA damage hypersensitivity of the H4T80A mutant was due to HR-mediated DNA repair defects, then mutation of H4T80A should not enhance rad52Δ phenotype. However, mutation of either H4R78A or H4T80A exacerbates the DNA damage hypersensitivity of rad52Δ cells  that has been previously shown to lower Rad53 activation levels  via its Tudor domain and γH2A via its BRCA1 C-terminal (BRCT) domains . InteresRTT107 mRNA levels do not significantly change upon CPT treatment and/or H4T80A mutation .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Prof. Tony Kouzarides . Unmodified and modified peptides columns were prepared by coupling either H4T80 or H4T80ph peptides to a sulfolink resin. Sera were first incubated with H4T80 column for 1 hour on rotation at room temperature. Supernatant was recovered and incubated with H4T80ph column for 1 hour on rotation at room temperature. H4T80ph column was then washed 6 times with TBS, and antibodies were eluted in 100mM Glycine pH 2.5. Elution was quickly neutralized with ice-cold 1M TrisHCl ph 8.8 solution, and dialyzed overnight in TBS + 10% glycerol.600 = 1. Cells were then collected, washed with water and frozen in liquid nitrogen. The cell pellet was resuspended in SP buffer  and spheroplasted by zymolyase digestion. Nuclei were then prepared by douncing in Lysis buffer  supplemented with both protease and phosphatase inhibitors (Roche). After spinning down in a benchtop centrifuge, supernatant was recovered and nuclei were pelleted by spinning at 20,000 rpm for 30 minutes in a Beckman SW-41 Ti rotor. Nuclei were then resuspended in NP buffer  supplemented with both protease and phosphatase inhibitors (Roche) and pelleted by spinning at 17,000 rpm for 30 minutes in a Beckman SW-41 Ti rotor. Nuclei were washed three times with Buffer A  supplemented with both protease and phosphatase inhibitors (Roche). Histones were then extracted in Buffer B  for 1 hour on rotation at 4°C and TCA precipitated.Yeast strains were cultured overnight to stationary phase. Next day, cells were grown in 1l of YPD for 3-4 generations to ODTotal protein extracts were prepared as previously described . Cells w600 = 0.5. Then, 2% galactose was added to induce expression of the HO endonuclease and 100 mL of yeast cultures per ChIP experiment were collected at the indicated times. Cells were cross-linked with 1% formaldehyde for 15 minutes at room temperature, and the reaction was quenched with 125mM glycine. Cells were resuspended in ChIP SDS buffer  supplemented with protease inhibitors (Roche) and disrupted with glass beads by using a FastPrep instrument . Chromatin was sonicated  to yield an average DNA fragment of 300-500 base pairs, and diluted 10 times in ChIP IP buffer  supplemented with protease inhibitors (Roche) prior to overnight immunoprecipitation on rotation at 4°C. Next day, 50μl of protein G dynabeads were added, and samples were incubated again on rotation at 4°C for 2 hours. Then, dynabeads were washed twice with the following buffers: TSE 150 , TSE 500  and LiCl buffer . DNA was eluted at 65°C in elution buffer , and cross-linking was reverted by overnight incubation at 65°C. Samples were treated with 0.5 mg/ml of RNase A at 37°C for 2 h. DNA was purified using the ChIP DNA Clean & Concentrator kit (Zymo Research). Relative DNA amounts were determined by qPCR using Fast SYBR Green Master Mix (Applied Biosystems). Primer pairs used for amplification are listed in the For the inducible DSB experiments, the corresponding yeast strains were grown in YP medium containing 2% raffinose until they reached ODPlasmids are listed in the 2; 1mM DTT; 150mM NaCl; 50mM β-glycerolphosphate; 0.05% [v/v] NP-40; 50mM cold ATP and 0.37MBq (10μCi ϒ32P-ATP), using 35 ng of GST-Cla4 or GST-Cla4K594A enzyme and 1μg calf H4, 4μg calf histone mixture , 2μg H4 peptide (residues 70-90) or 3μg of H4T80A peptide (residues 70-90) as substrates. Reactions on histones were separated by SDS-PAGE in 17% acrylamide gels, whereas reactions on peptides were resolved in 17% acrylamide Tricine gels. Gels were stained with Coomassie brilliant blue to visualize proteins and peptides, dried and exposed to film. Bands were quantified using ImageJ software. The mean values +SEM derived from two independent experiments were plotted using Prism .Kinase reactions were performed for 1h at 30°C in Kinase buffer  and poly-A mRNA was purified using poly-T oligo-attached magnetic beads (NEXTflex® Poly(A) Beads, catalog number NOVA-512981) according to the manufacturer’s instructions. RNA quantity and purity were assessed using an Agilent high-sensitivity RNA screen tape system  and Qubit . 200 ng of polyA were used to prepare libraries with the NEXTflex RNA-seq kit  according to the manufacturer’s instructions. Samples were barcoded and combined at uniform molarity to create a single pool, which was sequenced in a single-end 75-bp run on an Illumina NextSeq machine.For each strain, three independent colonies were grown in YPD medium to ODSaccharomyces cerevisiae genome using the STAR universal RNA seq aligner . RNA was purified using RNA Clean & Concentrator Kit , and cDNA was prepared using Superscript III reverse transcriptase . Expression levels of individual transcripts were determined by qPCR using Fast SYBR Green Master Mix  and oligonucleotides listed in the ACT1 mRNA in each sample. The mean values +SEM derived from the three biological replicates were plotted using Prism .For each strain, three independent colonies were grown in YPD medium to ODYeast cells were grown to exponential phase and whole cell extracts were prepared using glass beads in binding buffer  supplemented with both protease and phosphatase inhibitors. For peptide immunoprecipitation, 4μg of the corresponding peptide were diluted in 1ml of binding buffer and incubated with 75μl of Dynabeads MyOne Streptavidin C1 for 60 minutes at room temperature. Beads were washed twice with binding buffer. 0.5mg of whole cell extract was then incubated with beads for 3 hours at 4°C and washed 6 times with 300mM NaCl binding buffer. Immunoprecipitated proteins were then separated by SDS-PAGE in a 17% acrylamide gel.in vitro kinase assays. Microsoft excel software was used to perform all statistical analyses. Statistical differences were determined by two-tailed Student t test. Significance is denoted as ∗ for p < 0.05.ImageJ was used for quantification of The accesion number for the RNA-seq data reported in this paper is [ArrayExpress]: [E-MTAB-7090].Unprocessed imaging data are deposited on Mendeley Data:https://doi.org/10.17632/yjwrsb92fg.1


The structures of both epimers were elucidated from HRESIMS and 1D and 2D NMR data and the relative configuration of their macrolactone ring was assigned based on NOE and vicinal 1H NMR coupling constants and by calculation of a 3D model. Lanyamycin inhibited HCV infection into mammalian liver cells with an IC50 value of 11.8 µM, and exhibited a moderate cytotoxic activity against the mouse fibroblast cell line L929 and the human nasopharyngeal cell line KB3 with IC50 values of 3.1 and 1.5 μM, respectively, and also suppressed the growth of the Gram-positive bacterium Micrococcus luteus.Lanyamycin ( Sorangium cellulosum (strain Soce 481), we observed strong antifungal activity in the raw extract. On RP-HPLC fractionating and peak-activity correlation we further noted at tR = 18–25 min an inhibition overlap of Micrococcus luteus and Candida albicans, which correlated with some prominent peaks with unique HPLC-DAD-MS characteristics attracting our interest. Consequently, the fermentation process was optimized and scaled up. The main active principle was identified as soraphen A, which is already known from Sorangium cellulosum +, [M + Na]+, [M − H2O + H]+, [M − 2H2O + H]+, and [M − 3H2O + H]+ with high congruency of measured and calculated ion intensities while the [M + H]+ ion cluster was completely absent. In spite of their similarity, the lanyamycin epimers 1 and 2 could be separated by repeated RP-HPLC on Nucleodur Phenyl-Hexyl with an isocratic water/acetonitrile solvent system (52:48). Careful handling of the samples 1 and 2 allowed measuring 1D and 2D NMR data sets in methanol-d4 of both before a rearrangement finally restored the original mixture. Comparative NMR analyses showed that the spectra of 1 and 2 were very similar . The closure of the 16-membered lactone was apparent from the HMBC correlation between H-15 and C-1 (δC 166 ppm) and from the downfield shift of H-15 (δH 4.81 ppm). Together with four exchanged protons, the oxymethines  between δC 72 and 77 ppm were assigned as alcohol positions. The only remaining NH group finally filled the gap between the amide C-27 (δC 171 ppm) and C-26 (δC 75 ppm) based on the HMBC correlation H-26/C-27 thus forming the unusual and unstable N-acyl hemiaminal that explains the interconversion of lanyamycin isomers 1 and 2. The E configuration of the diene double bonds Δ10,11 and Δ12,13 was recognized from their large coupling constants of about 15 Hz while 1H,1H-ROESY correlations between H-3 and H-5 as well as between methyl-37 and both methyl-38 and methoxy-36 suggested the E configuration of the substituted Δ2,3,Δ4,5-diene. Likewise, the E configuration of the substituted Δ17,18 double bond was derived from the ROESY correlations between methyl-41 and methylene protons 16a and 16b and between methine-17 and methylenes H-20a and H-20b. The latter simultaneously indicated the s-trans configuration of the enone and explained the observation of a ROESY correlation between H-17 and H-21.RP-HPLC coupled to HRESIMS showed the two compounds were isomers with the elemental formula Cragments . Remarkaethyl-39 , Table 1d6 were measured  were identified as the NH and NH* signals of 1 and 2 from their correlation in the 15N,1H-HSQC NMR spectrum, respectively. They showed couplings to H-26 (4.96 ppm) and H-26* (4.91 ppm). The latter showed further 1H couplings to doublets of OH and OH* signals at 5.89 (5.6 Hz) and 5.95 ppm (5.4 Hz) and thus fully establishing the hemiacetal amide structural part. The mean value of the coupling constants between H-26 and H-25 remained as 5.7 Hz for 1 and 6.4 Hz for 2 indicating both compounds were epimers at C-26. Accordingly, 13C and 1H shift differences between lanyamycin epimers 1 and 2 mainly were observed in the side chains of the 16 membered macrolactone. As consequence of the epimerization, the conformation of the amide part of the side chain was adjusted. Particularly, the methyl group C-43 next to the epimeric centre was shifted from 0.87 ppm to higher field, i.e., 0.57 ppm, for C-43*. This allowed to observe a strong NOE between C-43 and 25OH, while the NOE between C-43* and 25*OH was only weak. Similarly, only the aromatic 35*/31* proton signals had an NOE with H-24* while the corresponding correlation was absent in isomer 1. Thus, the high-field shift (Δδ = 0.3 ppm) of C-43* was caused by the anisotropy of the aromatic ring, which requires a reorientation of the amide part of the hemiaminal in isomer 2.For confirmation, NMR spectra in DMSO-1/2 was found to be related to those characterizing the bafilomycins .Since the X-ray structure of bafilomycin Aanalyzed . These d1 and 2  and bafilomycins, including hygrolidins, micromonospolides and other bafilomycin type antibiotics, show very little similarity in their structures. The typical α-methyl-β-hydroxy part of the bafilomycin side chain is absent in 1/2, which features a unique α-methylene group. However, both share a carbonyl function in δ-position to the macrolide, which often is masked as a hemiketal in bafilomycins, e.g., in bafilomycin A1 D22 = +44° and [α]D22 = −136° , respectively; UV (methanol) λmax (log ε): 282 (4.407), 243 (4.821), 268 (4.335) nm; analytical RP-HPLC two peaks at 12.68 and 12.75 min with identical HRESIMS data: [2M + Na]+ calcd for C90H126N2NaO24, 1641.8592 (mσ 7.4); found, 1641.8607; [M + Na]+ calcd for C45H63NNaO12, 832.4242 (mσ 3.6); found, 832.4255 [M − H2O + H]+ calcd for C45H62NNaO11, 792.4317 (mσ 2.1); found, 792.4329; [M − 2H2O + H]+ calcd. for C45H60NNaO10, 774.4212 (mσ 117 due to overlap); found, 774.4228; [M − 3H2O + H]+ calcd for C45H58NNaO9, 756.4106 (mσ 8.8); found, 756.4120. NMR data in P ≤ 0.05. Viability assay results are given in 2 supply. Cells were infected with Jc1-derived Renilla reporter viruses in the presence or absence of compounds. Infected cells were lysed and then frozen at −80 °C for 1 hour following measurements of Renilla and Firefly luciferase activities on a Berthold Technologies Centro XS3 Microplate Luminometer as indicators of viral genome replication and cell viability, respectively, as previously described [Huh-7.5 cells were inoculated with RLuc Jc1 reporter viruses in the presence of different compounds. The inoculum was removed 4 hours later and monolayers were washed three times with PBS and overlaid with fresh medium containing no inhibitors. Infected cells were lysed 3 days later, and reporter virus infection was determined by renilla luciferase activity. The cell viability was measured by determination of firefly luciferase. The assay was performed in quadruplicate and is presented as the mean ± standard deviation. *** escribed .50, is the concentration of the compound that reduced the absorbance of the control sample by 50%. The cell culture was diluted to 50,000 cells per well. 120 µL of cell suspension was dispensed in every well in cell culture plate. Then a dilution plate was prepared by adding 100 µL media and 50 µL of test compound (1 mg/mL) in the first column of the plate. 100 µL medium was added into second to twelfth column wells. A serial dilution was done by transferring 50 µL from column 1 to column 2 cells and mixing twice, this was repeated all the way to the 12th column wells. Methanol was used as a negative control and epothilone-B as a positive control. The experiment was done in duplicate. 60 µL from the dilution plate was transferred to the respective well of the cell culture plate ending up with 180 µL of cell suspension plus diluted compound. The cell plate was incubated for 5 days at 37 °C and evaluated under a microscope for cell and nuclei morphological alterations regularly. 20 µL of MTT solution (5 mg/mL) was added to every well in the cell culture plate and incubated at 37 °C for 2–4 hours. The plate was centrifuged at 3000 rpm for 5 min, then 50–100 µL of PBS was added to each well. The plate was centrifuged again then the supernatant was discarded. 50–100 µL of isopropanol with 40 mM HCL was added to solubilize MTT crystals then the plate was shaken at 450–700 rpm on a plate shaker. The absorbance was measured at 595 nm to determine dead and living cells.The cytotoxicity was evaluated by measuring the effect produced on cell morphology including the nuclei and cell growth in vitro. Cell monolayers were prepared in 96-well microtiter plates and exposed to various concentrations of the compounds. The experiment was done in duplicate. Plates were checked by light microscopy after 24, 48 and 72 hours and on the 5th day, then cytotoxicity was scored as morphological alterations . The viability of the cells was determined by a tetrazolium-based colorimetric method using 3--2,5-diphenyltetrazolium bromide (MTT), as previously described . The 50%6 to 2.0 × 106 cells of test organism per mL measured using a calibrated photometer. The MIC values were determined in 96-well microtiter plates by 1:1 serial dilution in EBS medium  for bacteria and MYC medium , pH 7.0) for fungi as previously described [EBS or MYC media were diluted and standardized with microbial suspensions to about 1.0 × 10escribed . 150 µL escribed .File 11H and 13C NMR including the HCV infectivity results showing viability.Tables of NMR data and figures of the


Saturated fatty acids, such as palmitate, lead to circadian disruption. We aimed at studying the effect of low doses of palmitate on circadian metabolism and to decipher the mechanism by which fatty acids convey their effect in adipocytes. Mice were fed non-obesogenic doses of palm or olive oil and adipocytes were treated with palmitate and oleate. Cultured adipocytes treated with oleate showed increased AMPK activity and induced the expression of mitochondrial genes indicating increased fatty acid oxidation, while palmitate increased ACC activity and induced the expression of lipogenic genes, indicating increased fatty acid synthesis. Low doses of palmitate were sufficient to alter circadian rhythms, due to changes in the expression and/or activity of key metabolic proteins including GSK3β and AKT. Palmitate-induced AKT and GSK3β activation led to the phosphorylation of BMAL1 that resulted in low levels as well as high amplitude of circadian clock expression. In adipocytes, the detrimental metabolic alteration of palmitate manifests itself early on even at non-obesogenic levels. This is accompanied by modulating BMAL1 expression and phosphorylation levels, which lead to dampened clock gene expression. PERs and CRYs constitute part of the negative feedback loop that inhibits CLOCK:BMAL1-mediated transcription [The circadian clock, located in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus, responds to the environmental light–dark cycle and generates endogenous rhythms of ~24 h . Similarcription –8.Bmal1 expression [Bmal1 [Per2 null mice show altered lipid metabolism [The circadian clock regulates metabolism and energy homoeostasis in adipose tissues . This ispression  has beenpression . Deficiepression . In skelpression . Peroxisn [Bmal1  and circn [Bmal1 . Moreovetabolism . Glycogetabolism . Specifitabolism .High-fat diet (HFD) has been shown to influence clock oscillation and function in various animal studies –24. Thesin vitro in hepatocytes and in vivo in mouse liver. Oleate activated the AMPK–SIRT1 signalling pathway leading to inhibition of fatty acid synthesis and increased fatty acid oxidation, whereas palmitate activated mTOR signalling leading to increased fatty acid synthesis. This was achieved by modulating BMAL1 at several levels abrogating its activity and expression [The saturated fatty acid palmitate (C16:0) and the monounsaturated fatty acid oleate (C18:1), are the most common fatty acids in human diets as well as in animal and human fat tissue. While palmitate is associated with the development of obesity, and Type 2 diabetes, lipotoxicity and oxidative stress, oleate has been shown to prevent or alleviate the toxic effect of saturated free fatty acids and to be protective against insulin resistance and metabolic disorders . Palmitapression . In this2.2.1.ad libitum and then were randomly assigned to either olive oil (n = 24) or palm oil diet (n = 24) for 3 weeks. Control diet (n = 24) contained cornstarch 52.7% (w/w), casein 20% (w/w), sucrose 10% (w/w), soybean oil 7% (w/w), cellulose 5% (w/w), mineral mix 4% (w/w), vitamin mix 1% (w/w), methionine 0.3% (w/w). In palm and olive oil diet groups, soybean oil was replaced with olive oil  or palm oil . Body weight was recorded weekly and at the end of the experiment. Daily food intake was monitored every 2 days. Mice were placed on an overnight fast (12 h) before anaesthesia and epididymal adipose tissue was removed every 4 h (n = 4 for each time-point) around the circadian cycle in total darkness under a dim red light to avoid the masking effects of light. Tissue was immediately frozen in liquid nitrogen and stored at −80°C. Mice were humanely killed at the end of the experiment. Animals received humane care according to the criteria outlined in the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86–23 revised 1985). The joint ethics committee of the Hebrew University and Hadassah Medical Centre approved this study .Ten-week-old C57BL/6 male mice  were housed in a temperature- and humidity-controlled facility . Mice were entrained to 12 h light and 12 h darkness for 2 weeks with food available 2.2.2 at 37 °C. Differentiation of pre-adipocytes to adipocytes was achieved by allowing the cells to reach confluence. Two days later, the medium was replaced with DMEM supplemented with 10% FCS, 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 5 μg/mL insulin. Every 2 days thereafter, fresh medium (DMEM plus 10% FCS and 5 μg/mL insulin) was added to the culture for 8 days. On the 8th day of differentiation, cells were synchronized with a 1-h pulse of 1 mM dexamethasone  and medium was replaced with fresh DMEM supplemented with palmitate or oleate mixed in 1% bovine serum albumin (BSA) to a final concentration of 50 μM. For Control, medium was replaced with fresh DMEM containing 1% BSA. Following 6 h of incubation, cells were harvested in duplicates per treatment per time-point every 6 h for 24 h. Three independent experiments were performed with 2 replicates in each experiment.3T3-L1 pre-adipocytes were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% calf serum and 5% CO2.3.Lipid quantification in 3T3-L1 cells was performed using Oil Red O staining. Cells were fixed in 10% formaldehyde in aqueous phosphate buffer overnight, washed with 60% isopropanol and stained with Oil Red O solution (in 60% isopropanol) for 10 min. Cells were then repeatedly washed with water and destained in 100% isopropanol for 10 min. The optical density of the isopropanol solution was measured at 500 nm.2.4.(Table S1) spanning exon-exon boundaries and the ABI Prism 7300 Sequence Detection System . Gene expressing was normalized to actin. Reaction conditions were as follows: 3 min at 95°C, 10 sec at 95°C, 45 sec at 60°C. The fold change in target gene expression was calculated by the 2−ΔCt relative quantification method and by the 2−ΔΔCt for target gene oscillation. (Applied Biosystems).RNA of cells and tissue was extracted from cells using TRI Reagent (Sigma). Total RNA was DNase I-treated using RQ1 DNase  for 2 h at 37°C, as was previously described . Two μg 2.5.Cells were homogenized in lysis buffer , 50 mM sodium fluoride (NaF), 10 mM sodium orthovanadate (Na3VO4), 50 ng/ml aprotinin, 100 ng/ml leupeptin, 0.8 mg/ml trypsin inhibitor ) as was previously described . Samples2.6.p < 0.05. Statistical analysis was performed with JMP (version 14) software .All results are expressed as means ± SE. A one-way ANOVA (time of day) was performed to analyse circadian pattern of clock genes with several time-points. Tukey’s honestly significant difference (HSD) was performed as a single-step multiple comparison procedure in conjunction with ANOVA for the evaluation of significant differences among groups. A student’s t-test was performed for the evaluation of significant differences between two groups. Further analysis of circadian rhythmicity was performed using Circwave software (version 1.4) . For all analyses, the significance level was set at 3.3.1.Three weeks of palm or olive oil replacing soybean oil in the regular chow diet of mice did not affect food consumption or body weight . Similar3.2.Clock, Bmal1, Cry1, Per1, Per2 and Rorα compared to control , Cry1 , Per1 , Per2  and Rev-erbα  . Oleate treatment led to increased Clock  and Cry1  mRNA levels  ). In conA levels ). These 3.4.Pparγ  and Cebpα  and downregulated Fabp4 levels  and the mitochondrial genes Cox5b , ATPaseb2,  Ndufa2  and ND5 . In contrast, oleate significantly increased FABP4 mRNA levels  and the mitochondrial genes Cox5b , Ndufa2 , ND5 .  of palmitate or oleate. Palmitate upregulated the adipogenic genes t-test). ). These 3.5.To elucidate the metabolic pathways modulated by palmitate and oleate, we next assessed the daily average expression and/or activation levels of the key metabolic regulators AMPK, ACC, PP2A, GSK3β and AKT in 3T3-L1 adipocytes. Palmitate did not affect, while oleate significantly increased, the pAMPK/AMPK ratio compared to control  ). In con4.in vivo, in mice fed palm oil diet, and in cell culture, in 3T3-L1 adipocytes, with no effect of oleate. This finding is congruent with the reported effect of palmitate in mHypo-37 neuronal cells, inducing increased amplitude of Bmal1, which was lowered in the presence of DHA [Clock, Cry1, Per1, Per2 and Rev-erbα. Such observation is concordant with a recent report showing that palmitate suppressed clock gene expression [Bmal1 mRNA and protein levels.In this study, we demonstrated for the first time the effect of palmitate and oleate on the circadian clock in 3T3-L1 adipocytes. We show that non-obesogenic doses of palmitate were sufficient to alter circadian rhythms by increasing clock gene amplitudes both e of DHA . We havee of DHA . Althougpression . In contPparγ and Cebpα. Moreover, palmitate decreased the expression of the gene encoding the fatty acid chaperone FABP4, that triggers the ubiquitination and subsequent proteasomal degradation of PPARγ [Atpase2b, Cox5b, Ndufa2, and Nd5. In contrast, oleate induced the expression of the gene encoding FABP4, the expression of genes encoding the aforementioned electron transport chain and ATP synthesis proteins and Cpt1 mRNA levels. Thus, palmitate increases signalling towards lipogenesis and decreases signalling towards fatty acid oxidation, while oleate increases catabolic signalling pathways. These metabolic findings are concordant with our previous report showing the effect of oleate and palmitate on mouse liver and hepatocytes [BMAL1 has been reported to regulate lipid accumulation in adipose tissue ,12. Its of PPARγ . In addiatocytes .AMPK is a key metabolic factor that regulates circadian clock . While pIn summary, in adipocytes, oleate activates the AMPK signalling pathway leading to inhibition of fatty acid synthesis and increased fatty acid oxidation. In contrast, palmitate activates ACC pathway leading to increased fatty acid synthesis. This is achieved by inducing the activity of PP2A. Induced PP2A activity increases GSK3β activity and, as a result AKT activity, leading to the phosphorylation of BMAL1, which leads to adipogenesis and lipogenesis and high amplitude but dampened clock gene expression. The intricate relationship between core clock proteins and fatty acids warrants further study.


Currently, a common therapeutic approach in cancer treatment encompasses a drug combination to attain an overall better efficacy. Unfortunately, it leads to a higher incidence of severe side effects, namely cardiotoxicity. This work aimed to assess the cytotoxicity of doxorubicin , 5-fluorouracil (5-FU), cyclophosphamide (CYA), and their combination  in H9c2 cardiac cells, for a better understanding of the contribution of each drug to FAC-induced cardiotoxicity. Differentiated H9c2 cells were exposed to pharmacological relevant concentrations of DOX (0.13–5 μM), 5-FU (0.13–5 μM), CYA (0.13–5 μM) for 24 or 48 h. Cells were also exposed to FAC mixtures . DOX was the most cytotoxic drug, followed by 5-FU and lastly CYA in both cytotoxicity assays -2,5-diphenyl tetrazolium bromide (MTT) and neutral red (NR) uptake). Concerning the equimolar combination with 1 or 5 μM, FAC caused similar cytotoxicity to DOX alone. Even in the presence of higher concentrations of 5-FU and CYA (50 μM 5-FU + 1 μM DOX + 50 μM CYA), 1 μM DOX was still a determinant for the cardiotoxicity observed in the cytotoxicity assays, phase contrast morphological evaluation, and mitochondrial potential depolarization evaluation. To the best of our knowledge, this was the first in vitro work with this combination regimen, DOX being the most toxic drug and key to the toxicity of FAC. Cancer is one of the leading causes of death worldwide. Although the number of diagnosed cases is increasing, overall cancer prognosis and 5-year survival is rising in the majority of cancer types. Early diagnosis and the extended therapeutic alternatives and strategies that exist nowadays contribute to this reality. New drugs are emerging for cancer treatments with good results, but combined chemotherapy is still the most common and well characterized option in several cancers ,2,3. ComIn Europe, breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among women . WorldwiCardiotoxicity is a common side effect of anticancer drugs ,10,11. DAll plastic sterile material used in the cell culture was purchased to Corning-Costar . Phosphate-buffered saline (PBS) without calcium and magnesium and penicillin/streptomycin were obtained from Biochrom . Fetal bovine serum (FBS), Hanks’ balanced salt solution (HBSS) and Dulbecco’s phosphate buffered saline (PBS) with calcium and magnesium were acquired to Gibco . Other compounds namely DOX hydrochloride, CYA, 5-FU, trypan blue solution (0.4%), Dulbecco’s modified eagle medium (DMEM) high glucose, sodium bicarbonate, dimethyl sulfoxide (DMSO), retinoic acid (RA), trypan blue solution (0.4%), neutral red (NR) solution, trypsin solution, 3--2,5-diphenyl tetrazolium bromide (MTT), sodium dodecyl sulfate (SDS), Hoechst 33258 solution, and paraformaldehyde were obtained from Sigma-Aldrich . The cytotoxicity of DOX, CYA, 5-FU and of their equimolar FAC mixtures  was assessed in differentiated H9c2 cells. Besides, a FAC mixture with concentrations based in the plasma levels of treated patients   was also2. All experiments were carried out before the cells reached 70–80% confluence and the cell line was used between passage 7 and 29 [per se and, therefore, the cells were exposed to each drug. Control wells were exposed to PBS in the same volume of treated cells.The rat cardiomyocyte derived H9c2 cell line was obtained from the European Collection of Cell Cultures (H9c2 cell line from rat (BDIX heart myoblast), from Sigma-Aldrich ). H9c2 cells were obtained from the ventricular part of a thirteenth-day rat heart embryo  and were7 and 29 . H9c2 ce7 and 29 ,20,21. C7 and 29 . All dru-per se in different concentrations (DOX (0.13–5 µM), 5-FU (0.13–5 µM), CYA (0.13–5 µM));the drugs -equimolar mixtures of the parent drugs );-FAC mixture with concentrations based in the plasma levels of treated patients (50 µM 5-FU + 1 µM DOX + 50 µM CYA) .After the 7-day differentiation protocol described, H9c2 cells were exposed to:Two cytotoxicity tests were done. Moreover, morphological evaluation, using phase contrast microscopy and Hoechst nuclear staining, was performed. Mitochondrial membrane potential was also assessed with the parent drugs and their combination (50 µM 5-FU + 1 µM DOX + 50 µM CYA). All these determinations will be described below.per se were tested for 24 or 48 h, while the mixtures, FAC, were tested at 48 h. Next, the media was removed and the protocol was done according to a previously described procedure [The mitochondrial dehydrogenases and other reducing agents and enzymes located in other organelles reduce the water-soluble yellow tetrazolium dye, MTT, into an insoluble blue formazan that can be measured at 550 nm. The drugs rocedure , in whicper se for 24 or 48 h, whereas the effects on NR uptake assay of the mixtures, FAC, were assessed at 48 h since the majority of the cardiotoxicity observed after chemotherapy is late stage. At the end of the incubation period, cells were exposed to the differentiation medium enriched with 33 μg/mL of NR for 3 h at 37 °C. Then, the cells were washed with warm HBSS with calcium and magnesium and the NR within the cells was released adding an ethanol: acetic acid solution (50%: 1% v/v) in water. After a 15-min agitation period, the absorbance was measured at 540 nm and 690 nm [The amount of NR dye incorporated into the cells represents their lysosomal functionality, as this dye easily enters cell membranes and is stored in lysosomes . The celd 690 nm , in a muper se by phase contrast microscopy, using a Nikon Eclipse TS100 equipped with a Nikon DS-Fi1 camera . At 48 h, the morphology of the cells was evaluated in all possible drug combinations and respective drugs Hoechst staining was used to assess the effect of the drugs or their mixture on nuclear morphology of differentiated H9c2 cells. After a 48-h exposure, cells were fixed with 4% paraformaldehyde solution  and washed 3X with PBS containing calcium and magnesium. Cells were then stained with the nuclear dye Hoechst 33258  for 10 min at 37 °C (protected from light). The cells were washed with PBS containing calcium and magnesium (3X) and then observed in a Nikon Eclipse TS100 equipped with a Nikon DS-Fi1 camera using a fluorescent filter (λ excitation maximum = 346 nm and λ emission maximum = 460 nm) . 2 incubator at 37 °C for 15 min (protected from light). In sequence, the 48-well plates were centrifuged for five min at 400× g at room temperature and the medium was removed carefully. In each well, 250 µL warm HBSS with calcium and magnesium were added and the plate was centrifuged for additional five min at 400× g at room temperature to remove the excess probe, being the supernatant aspirated carefully. This washing step was repeated three times. Fluorescence was read at a λ excitation maximum = 485 nm and a λ emission maximum = 535 nm  and at a λ excitation maximum = 535 nm and a λ emission maximum = 595 nm  in a multi-well plate reader ). The cells were exposed for 48 h to the drugs or their combination to assess their influence on mitochondrial membrane potential. Then, JC-1  was added to each well. One hundred µM of the protonophore, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), was used as an uncoupler of mitochondrial oxidative phosphorylation. The cells were placed in a COpost-hoc assay when a significant p was reached. When the distribution was normal, a parametric analysis of variance (ANOVA) was performed, followed by the Tukey’s post hoc test. Statistical significance was reached when p < 0.05. All statistical analyses were performed in GraphPad Prism 7 software .Data are expressed as mean ± standard deviation (SD). The outliers were evaluated by the Robust regression and Outlier removal (ROUT) test. The D’Agostino & Pearson normality assay was used to evaluate data distribution. When data did not follow a normal distribution, statistical analysis was performed using the Kruskal–Wallis test, followed by the Dunn’s The MTT reduction assay was done at two time-points (24 and 48 h) using several DOX concentrations (0.13–5 μM). Although at 24 h, DOX caused small but significant changes when compared to control cells A, those The lysosomal uptake of NR was also evaluated after DOX exposure at two time-points. The three highest tested concentrations of DOX  caused a significant impairment of lysosomal NR uptake when compared to control cells at 24 h C. That cThe MTT reduction assay was assessed at two time-points (24 and 48 h) using several 5-FU concentrations (0.13–5 μM). At 24 h, no important cytotoxicity was observed for 5-FU, except for 5 µM A. At 48 The NR lysosomal uptake assay was also performed after 24-h or 48-h exposures to 5-FU (0.13–5 μM). Only at the highest concentrations of 5-FU  a small, but statistically, impairment of lysosomal NR uptake was found when compared to control cells at 24 h C. At 48 The CYA effects on the MTT reduction and the lysosomal NR uptake were evaluated at two time-points (24 and 48 h) using several CYA concentrations (0.13–5 μM). At 24 h, CYA 1 µM caused a significant cytotoxicity when compared to control cells, but no changes were seen in other CYA concentrations when compared to control cells A. At 48 per se  was tested in differentiated H9c2 cells. In the MTT reduction assay, the FAC mixture caused cytotoxicity at the concentrations of 0.2 µM (87.14 ± 7.05%), 1 µM (76.12 ± 9.07%) and 5 μM (70.06 ± 9.33%), in a concentration-dependent manner. The mixture of DOX+5-FU and DOX+CYA also produced a significant decrease in MTT reduction ability, after a 48-h exposure to 0.2, 1 or 5 μM concentrations . FAC ledper se A.After a 48-h exposure to the equimolar FAC mixture at the concentration 0.2, 1 or 5 μM (each drug), the lysosomal NR uptake assay was performed. FAC mixture caused cytotoxicity at the concentrations of 0.2 µM (87.62 ± 8.95%), 1 µM (50.03 ± 14.52%) and 5 μM (28.40 ± 4.24%), in a concentration dependent manner. No significant differences were found between FAC and DOX, at any of the tested conditions D–F.Regarding phase contrast microscopy at 48 h, 1 µM DOX caused cellular damage, with evident cellular debris, with round and detached cells . There wIn the Hoechst staining, no nuclear condensation was seen in any of the conditions evaluated . The MTT reduction assay was done at 48 h to evaluate the effect of the FAC mixture at concentrations based in the plasma levels of treated patients . This FAPhase contrast microscopy evaluation at 48 h of the FAC combination based in the plasma levels of treated patients showed a small increase in cytotoxicity, with higher cell detachment and the number of round cells, when compared to DOX 1 µM alone . MoreoveJC-1 staining showed a significant decrease in mitochondrial membrane potential of cells exposed to all mixtures containing DOX and to DOX alone when compared to the control . The mitThis work, to the best of our knowledge, is the first to assess in vitro the cardiotoxicity of several combinations of FAC (5-FU + DOX + CYA) in differentiated H9c2 cardiac cells and to test such a large range of drug concentrations. The major findings of this work were: (i) DOX caused a time-dependent toxicity on both cytotoxicity assays performed (MTT reduction and NR uptake); (ii) 5-FU was only consistently toxic at the highest concentrations tested (5 and 50 µM), whereas CYA had the lowest toxicity in these experimental conditions; (iii) the equimolar combinations of FAC had similar toxicity when compared to DOX alone, except at the 0.2 µM mixture, while FAC caused higher toxicity than 0.2 µM DOX alone; (iv) the clinically relevant FAC mixture (50 µM 5-FU + 1 µM DOX + 50 µM CYA) had a similar toxicity when compared to 1 µM DOX, in the MTT reduction and NR uptake assays and mitochondrial membrane potential evaluation, although phase contrast microscopy showed a higher toxicity of that combination.After the differentiation process, the cells were exposed to DOX at concentrations that ranged from 0.13 to 5 μM. According to the MTT reduction test, all tested concentrations caused toxicity to the differentiated H9c2 cells and DOX had the highest toxicity when compared to the other tested drugs. At 24 h, 5 µM DOX caused approximately a 10% decrease in MTT reduction, whereas 0.13 and 1 µM caused a decrease of about 5%, when compared to control values. In the work of Green and Leeuwenburgh, the authors tested the cytotoxicity of DOX  in non-differentiated H9c2 cells by the MTT reduction test after a 20 h exposure . These dIn our work, the cytotoxicity-induced by DOX was higher in the NR uptake assay than in the MTT reduction assay. To the best of our knowledge, there are no studies with DOX on differentiated H9c2 cells using the NR uptake assay. The higher sensitivity to DOX at the highest concentrations tested can be due to autophagy induction involving lysosomes ,29. TherRegarding 5-FU, only the highest concentrations caused a slight but significant cytotoxicity in differentiated H9c2 cells. Accordingly, Lamberti and collaborators showed that 5-FU is substantially less toxic in non-differentiated H9c2 cells than DOX  . Finally2, 18% when the dose is 551–600 mg/m2, and 36% when the dose exceeds 600 mg/m2 [2, 26% at 550 mg/m2, and 48% at 700 mg/m2 [As stated in the introduction section, the FAC combination led to huge increases in disease-free survival in breast cancer patients ,14,31. N00 mg/m2 . Other v00 mg/m2 . Other r00 mg/m2 . Neverth00 mg/m2 ,17.th dose, the levels are lower than 205 µM, possibly due to enzymatic metabolic auto induction [2 DOX over 15 min followed by 600 mg/m2 CYA over 15 min and the maximum observed plasma of CYA concentrations were about 100 µM [2 DOX [In cancer patients, DOX plasma levels may be found in the range of 0.4 µM ,36 but anduction . Sixty-ft 100 µM . The widM [2 DOX . Our stu2 docetaxel and 600 mg/m2 CYA, did not increase the risk for cardiotoxicity when compared to anthracycline-containing regimens, such as the combination of 60 mg/m2 DOX and 600 mg/m2 CYA [When facing these results, we evaluated the effects in mitochondrial membrane potential of the FAC mixture (50 µM 5-FU + 1 µM DOX + 50 µM CYA). DOX is a known mitochondrial toxicant  and we ag/m2 CYA . Accordig/m2 CYA . Moreoveg/m2 CYA ,46,47,49g/m2 CYA ,15,16,17g/m2 CYA .In conclusion, this is a pioneer work that shows in vitro that DOX is the major toxicological determinant for the cardiotoxicity of FAC mixture. According to the studies presented, DOX seems to be the drug that mostly contributes to the cardiotoxicity of the FAC mixture in clinical studies, as it happens in this work, demonstrating that this mixture paradigm and cellular model can be attractive to determine the mechanisms of toxicity of the usual chemotherapeutic regimens.


The interaction between human epididymis protein 4 (HE4) and annexin A2 (Annexin A2) has been found in ovarian cancer. However, it is dimness whetherthe interaction exists in other malignant tumors.Real-time PCR, western blotting and immunocytochemistry were used to detect mRNA and proteins expression. Co-immunoprecipitation and double-labeling immunofluorescence were used to detect the interaction among HE4, ANXA2 and MMP2. MTS assay was used to test cell proliferation. Adhesion test was used to test cell adhesion. Flow cytometry was applied to examine cell cycle. The scratch test and Transwell assay was performed to detect the migration and invasion of various malignant cell lines.Here we show that the overexpression of HE4 and ANXA2 in various malignant cells is a common phenomenon. HE4 and ANXA2 are co-localized in the cytoplasm and membrane of various tumor cells. ES-2 cells which had both high expression of HE4 and ANXA2 were much stronger in proliferation, adhesion, invasion, and migration than other tumor cells. HE4–ANXA2–MMP2 could form a triple protein complex. HE4 could mediate the expression of MMP2 via ANXA2 to promote cell migration progress.The interaction of HE4 and ANXA2 exists in various types of cancer cells. HE4 and ANXA2 can promote the proliferation, adhesion, invasion, and migration of cancer cells. HE4–ANXA2–MMP2 form a protein complex and ANXA2 plays the role of “bridge”. They performed together to promote cell migration. Human epididymis protein 4 (HE4), also known as WFDC2, was discovered in 1991 and named because of its specific expression in epithelial cells of the epididymis  completeIn protein mass spectrometry, we found a HE4 interacting protein, annexin A2 (ANXA2). HE4/ANXA2 interaction was confirmed by GST pull-down and co immunoprecipitation. HE4/ANXA2 promoted the invasion and migration of ovarian cancer cells. In the past 24 years since the discovery of HE4, There’s been amount of research into the HE4 clinical application, but little is known about its biological functions and mechanisms. Our preliminary studies have shown that the proliferation, invasion, migration, drug resistance and other functions of ovarian cancer cells increased after upregulate of HE4 expression, or after treated with HE4 active protein –10. ANXAAccording to our preliminary studies, ANXA2 is highly expressed in ovarian  and endo2/95% air atmosphere and were revived every 3 to 4 months.An ovarian cancer cell line (CaoV-3、ES-2、OVCAR3), endometrial carcinoma cell lines (Ishikawa), lung cancer cell line (A549), cervical cancer cell line (Hela), hepatic carcinoma cell line (HepG2), gastric cancer cell line (HGC-27), colon carcinoma cell lines  and human embryo kidney cell line (HEK293) were cultured according to the manufacturer’s protocol. All of the cell lines used in the experiment were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. ES-2 was propagated in McCoy’s 5A modified medium with 10% fetal bovine serum; HepG2, HEK293 were propagated in DMEM high glucose medium with 10% fetal bovine serum; other cell lines were cultured in RPMI-1640 medium with 10% fetal bovine serum. All the cell lines were grown at 37 °C in a 5% COTransfection reagent kits are purchased from invitrogen reagent company. pEGFP-N1-ANXA2 plasmids are purchased from Shanghai GenecChem, and according to the plasmid transfection specifications, this plasmid is transfected into HEK293 cells and stably construct cell line 293-A2. The lentivirus-mediated HE4 shRNA sequence is: 5′-GTCCTGTGTCACTCCCAAT-3′, and the control sequence 5′- TTCTCCGAACGTGTCACGT -3′. The targeting sequences were cloned into GV248 vector (Shanghai GeneChem). The depletion efficiency was evaluated by western blot analysis.7 cells) to extract total RNA. cDNA was synthesized according to the RNA reverse transcription kit instructions . The sequences of HE4 gene primer were 5′-TGCCCCCAGGTGAACATTAAC-3′ for forward primer and 5′-CCATTGCGGCAGCATTTCAT-3′ for reverse primer. The sequences of ANXA2 gene primers were 5′-CTGCTCCAGAACCAACCAG-3′ for forward primer and 5′-TGCGGAAGTCACCAGATGT-3′ for reverse primer. The sequences of GAPDH primers were 5′-ATGGAAATCCCATCACCATCTT-3′ for forward primer and 5′-CGCCCCACTTGATTTTGG-3′ for reverse primer. The Light Cycler PCR system  was used for real-time PCR amplification and Ct value detection. The melting curves were analyzed after amplification. PCR reactions of each sample were done in triplicate. Data were analyzed through the comparative threshold cycle (CT) method.Cells were added with Trizol reagent  and 1:300 , respectively. The primary antibody was replaced by rabbit IgG for negative control.g for 30 min at 4 °C, the supernatant was collected and treated with 10 μl of mouse anti-ANXA2 monoclonal  or goat anti-HE4 polyclonal antibody  for 3 h at 4 °C. Then, 20 μl of protein A/G PLUS-Agarose  was added, followed by incubation on a rocker platform overnight at 4 °C. The primary antibody was replaced by mouse or goat IgG  as negative control. Immunoprecipitates were subsequently subjected to 12% SDS gel electrophoresis and analyzed via western blot using rabbit polyclonal HE4  and mouse monoclonal ANXA2 antibodies . Proteins were visualized using ECL reagent (Thermo scientific ECL). The experiments were repeated three times.Ice-cold RIPA buffer (1 ml) was added to cells, followed by incubation for 30 min at 4 °C. After centrifugation at 15,000×Cells in the exponential phase of growth were digested with 0.25% trypsin and cultured in medium containing 10% FBS to prepare single-cell suspension. Cells were washed twice with cold PBS when growing in a single layer, and fixed with 4% para-formaldehyde for 30 min. The cells were simultaneously incubated with primary antibodies against HE4  and ANXA2 . The primary antibody was replaced by rabbit or mouse IgG for negative controls. The working concentrations of fluorescein isothiocyanate (FITC) and tetraethyl rhodamine isothiocyanate (TRITC) were 1:50. Nuclei were counterstained with DAPI. The empirical procedure was performed according to the manufacturer’s instructions.Cells during the log phase were selected and seeded into 6-well plates. When cell confluence was 90%, the cells were starved with serum-free medium overnight. Then scrathed the plate straightly with 10 µl pipette. Cells were cultured in medium without serum. After 24 h, the width of the scarification were observed via inverted microscope  with 100× magnification. Wound-healing percentage of the cells was determined by the ratio of healing width at each time point to the wound width at 0 h.5 cells were seeded in 200 μl serum-free medium in the upper chamber coated with matrigel . The pipette tip was pre-cooled, and the ECM gel was melted at 4 °C overnight, diluted by 1:8 with serum free medium. Complete medium was added to the lower chamber. After 24 h or 48 h incubation at 37 °C, cells in the upper chamber were carefully removed with a cotton swab and the cells traversed to the back membrane of the chamber were fixed in 4% para-formaldehyde and stained with 0.1% crystal violet. For quantification, five fields  per filter were counted under a microscope .Cell invasion assay were performed using transwell chamber with pore size of 8 μm. For the invasion assay, 2 × 10R AQueous One Solution Cell Proliferation Assay . Cells were seeded at a density of 103 per well in 96-well culture plate. Cell proliferation was detected at 24, 48, 72 h, respectively. 100 μl medium were added with 20 μl MTS, followed by incubation for 2 h. The optical density at 490 nm was measured on an ELX-800 spectrometer reader .Cellular proliferation was analyzed by CellTiter 96Cells were harvested and washed with PBS for three times. Then, they were fixed with 70% cold ethanol at 4 °C overnight. Cells were resuspended and washed with PBS, then resuspended in 100 μl RNase, 37 °C for 30 min. Then added 400 μl propidium iodide (PI), 4 °C in dark for 30 min, and quantified by FACSCalibur . The empirical procedure was performed according to the manufacturer’s instructions and repeated for three times.3 per well and incubated at 37 °C for 10, 30, 60 min, then washed with PBS to remove non-adherent cells. Adherent cells were fixed with 4% para-formaldehyde, after washed by PBS, cells were stained with crystal violet. Washed with PBS and added 2% SDS to terminate the reaction. The optical density at 570 nm was measured on an ELX-800 spectrometer reader .Cells were seeded in 96-well plates at a density of 2 × 102 analysis, variance analysis, and t test were employed. p < 0.05 was considered statistically significant.SPSS version 17.0  software was used for statistical analysis. χWe detected protein and mRNA expression levels of HE4 and ANXA2 in nine types of malignant cells lines  and Human Embryonic Kidney 293 cell line by western blot Fig. a, immunoWe further studied the HE4-ANXA2 interaction in different cell lines by co-immunoprecipitation and double-label immunofluorescence assays. Co-immunoprecipitation  . Both ES-2 and HT29 cell lines are highly expressed of ANXA2, which indicated that ANXA2 may play an important role in the proliferation of malignant cells  Fig. a. The prlls Fig. b.Fig. 4Cp < 0.05). ES-2 has high expression of both HE4 and ANXA2, which indicated that a concomitant high expression of HE4 and ANXA2 might promote the adhesion behavior of tumor cells . HE4 expression level of ES-2 was higher than that of HGC-27 and HT29, which both cell lines had relatively low expressing of HE4. The above results suggested that HE4 might play an important role in invasion and metastasis of tumor cells (Fig. The migration ability and invasive ability of the cells were detected by scratch test, matrigel-transwell chamber assay. The results showed that migration and invasion ability of ES-2 was significantly stronger than those of HGC-27 and HT29 (Co-immunoprecipitation was used to detecte the interaction between HE4/ANXA2 and MMP2. The results showed interaction between HE4/ANXA2 and MMP2 was common in various malignant tumor cells including CaoV3, ES-2, OVCAR-3, Ishikawa, A549, HGC-27, SW480, HepG2, Hela, HT29 and HEK293 cell lines (Fig. The result of western blot and transwell migration experiments demonstrated the expression of ANXA2, MMP2 and ability of cell migration decreased when HE4 gene was silenced in HEK293 and CaoV3 cell lines, and recovered after adding human recombinant ANXA2 active protein Fig. c–e, g. AWFDC2 gene and contains two WAP domains; it is considered to be homologous to the extracellular proteinase inhibitor, SLPI protein because of its structural specificity. HE4 has been found in the epithelial cells of the distal spermatic duct and may be involved in sperm maturation [HE4 protein is encoded by the turation . Subsequturation , which hturation . The bioturation  showed tturation  found, uturation  found, ituration  further turation  found, tturation  demonstrturation  showed tIn response to the controversies surrounding the biological functions of HE4 in ovarian cancer cells, our research group previously examined these biological functions in in vivo and in vitro models by transfection and silencing of HE4, and by adding HE4 active protein to cultured cells , 26, 27.ANXA2 is a HE4-interacting protein in ovarian cancer which was discovered by our research group through mass spectrum analysis in the preliminary phase . ANXA2, In order to further substantiate whether HE4 and ANXA2 interaction is ubiquitous in malignant tumor cells, we detected the expression and interaction of HE4 and ANXA2 in different malignant tumor cell lines, and then compared the differences between the proliferation, invasion, migration and adhesion of cell lines with different expressions level of HE4 and ANXA2. The results showed that HE4 and ANXA2, both located in the membrane and cytoplasm, were highly expressed and widely interacted in various malignant tumor cells. Cell lines that expressed high levels of HE4 and ANXA2 showed the greatest ability in invasion, migration, proliferation, and adhesion. Cell lines with high level of ANXA2 showed higher proliferation ability.MMP2, is a matrix hydrolase that specifically dissolves IV collagen in matrix metalloproteinase family; Its expression is positively correlated with tumor progression, metastasis, and poor prognosis , 40. A sIn conclusion, this study has revealed that HE4 is expressed in several malignant tumor types where it interacts with ANXA2 to facilitate invasion, migration, adhesion, and proliferation. HE4, ANXA2 and MMP2 can form a protein complex. HE4 also promotes cellular invasion and migration through influencing MMP2 expression via ANXA2. These findings provides an important experimental basis for the biological function of HE4 and ANXA2 in the progression of malignant tumors.In summary, the interaction of HE4 and ANXA2 commonly exists in various cancer cells. HE4 and ANXA2 can synergistic promote the proliferation, adhesion, invasion, and migration of cancer cells. In addition, HE4 and MMP2 form a protein complex and ANXA2 plays the role of “bridge”. They work together to promote cell migration.