The B7 family member, B7H6, is a ligand for the natural killer cell receptor NKp30. B7H6 is hardly expressed on normal tissues, but undergoes upregulation on different types of tumors, implicating it as an attractive target for cancer immunotherapy. The molecular mechanisms that control B7H6 expression are poorly understood. We report that in contrast to other NK cell ligands, endoplasmic reticulum (ER) stress upregulates B7H6 mRNA levels and surface expression. B7H6 induction by ER stress requires protein kinase R-like ER kinase (PERK), one of the three canonical sensors of the unfolded protein response. PERK phosphorylates eIF2α, which regulates protein synthesis and gene expression. Because eIF2α is phosphorylated by several kinases following different stress conditions, the program downstream to eIF2α phosphorylation is called the integrated stress response (ISR). Several drugs were reported to promote the ISR. Nelfinavir and lopinavir, two clinically approved HIV protease inhibitors, promote eIF2α phosphorylation by different mechanisms. We show that nelfinavir and lopinavir sustainably instigate B7H6 expression at their pharmacologically relevant concentrations. As such, ER stress and ISR conditions sensitize melanoma targets to CAR-T cells directed against B7H6. Our study highlights a novel mechanism to induce B7H6 expression and suggests a pharmacological approach to improve B7H6-directed immunotherapy.B7H6 is induced by ER stress in a PERK-dependent mechanism.Induction of B7H6 is obtained pharmacologically by HIV protease inhibitors.Exposure of tumor cells to the HIV protease inhibitor nelfinavir improves the recognition by B7H6-directed CAR-T.The online version of this article (10.1007/s00109-019-01859-w) contains supplementary material, which is available to authorized users. Natural killer (NK) cells are effector lymphocytes of the innate immune system. They are able to recognize and lyse tumor and virus-infected cells without priming. Their activity is governed by a delicate balance between activating and inhibitory receptors. Among the activating receptors are the natural cytotoxicity receptors (NCRs), a family of three members: NKp30, NKp44, and NKp46. These receptors bind a diverse array of cellular- and pathogen-associated ligands, and they induce the killing activity of NK cells , 2.B7 homolog 6 (B7H6), also known as NCR3LG1 , is a human-specific B7 family member that binds to and activates the NKp30 receptor. B7H6 is typically not expressed on normal human tissues, but it is expressed on approximately 20% of human tumor cell lines including melanoma, carcinomas, T and B lymphomas, and myeloid leukemias, as well as primary tumor blood and bone marrow cells . BesidesThe endoplasmic reticulum (ER) is the first compartment of the secretory pathway, in which proteins destined to other organelles are folded and integrated into the membrane. Conditions of ER stress develop following the accumulation of improperly or partially folded proteins in the ER or other perturbations to ER homeostasis. Signaling pathways are activated upon ER stress in an attempt to restore ER homeostasis, collectively termed the unfolded protein response (UPR). The UPR is triggered by three key transmembrane proteins: inositol-requiring enzyme 1 (IRE1), protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6). Upon accumulation of improperly folded proteins in the ER, IRE1 and PERK are activated by oligomerization and trans-autophosphorylation. IRE1 contributes to the unconventional splicing of the mRNA of the transcription factor X-box binding protein 1 (XBP1) to produce its active and more stable form of spliced XBP1 (XBP1s), which activates the transcription of UPR target genes . PERK isThe UPR plays important roles in the development and function of the immune system , 21. It 2.The human melanoma cell lines 624 and 526 were provided by Dr. Michal Lotem . Cells were cultured in Dulbecco’s modified Eagle medium  supplemented with 10% fetal bovine serum , 2 mM L-glutamine , and 1% penicillin-streptomycin and sodium pyruvate solutions  at 37 °C under 5% COHuman peripheral blood mononuclear cells (PBMCs) were isolated from whole blood or leukapheresis of healthy donors by a ficoll density gradient and activated for 2 days with OKT-3 (Miltenyi) in the presence of IL-2 (Miltenyi). Activated cells were then transduced with a retroviral vector coding for the anti-B7H6 CAR fused to a Furin T2A cleaving site in frame with a truncated human CD19 marker (tCD19) described in  or a vecThapsigargin , GSK2606414 , cycloheximide , ISRIB , nelfinavir , lopinavir , ionomycin , PMA . Stock solutions of Tg, GSK, ISRIB, Nel, and Lop were prepared in DMSO at concentrations of 2.5 mg/ml, 1 mM, 0.5 mM, 5 mM, and 10 mM, respectively. CHX was dissolved in ethanol at 50 mg/ml concentration. The final concentration of each compound was 0.125 μg/ml, 1 μM, 0.5 μM, 10 μM, 20 μM, and 100 μg/ml, respectively. The corresponding solvent was used as a vehicle control for each treatment.Knockout cells were produced as previously described . In brie3). The cells were then incubated for 30 min on ice with anti-mouse AlexaFluor 647 secondary antibody (Jackson ImmunoResearch). The following primary antibodies were used: anti-MICA , anti-MICB , anti-ULBP1 , anti-ULBP2/5/6 , anti-ULBP3 , anti-B7H6 , anti-PVR (in-house developed), anti-HLA1 (W6/32), anti-Beta-2 microglobulin , anti-Ceacam-1 , anti-Nectin-2 . Mouse IgG1 , IgG2a , and IgG2b  were used as an isotype control.Melanoma 624 wt or knockout cells were plated at equal densities and incubated overnight. Resuspended cells were incubated on ice for 1 h with the primary antibody at a concentration of 0.2 μg/well in FACS buffer  was generated by de novo total synthesis  and cloned into a lentiviral vector. Then, using site-directed mutagenesis, a T in the upstream ATG of the 5′UTR was replaced by A (AAG-5′BG) Fig. . A PCR r2, 1 mM dithiothreitol) supplemented with 1% triton, 30 U/ml Turbo DNase (Ambion), and 100 μg/ml cycloheximide in DEPC water. The lysed samples were centrifuged at 12,000g at 4 °C for 10 min. The cleared lysates were loaded onto a 10–50% sucrose gradient and centrifuged at 35,000 rpm in an SW41 rotor for 3 h at 4 °C. Gradients were fractionated into 12 fractions, and the optical density at 254 nm was continuously recorded using a Biocomp gradient station. The fractions were combined into three phases, polysome free, light, and heavy depending on the UV reading. The amount of B7H6 mRNA was determined in each phase by qRT-PCR.Polysome profile analysis was carried out as described above . Briefly5 cells of each treatment were collected and cocultured with B7H6-specific CAR T cells in round-bottom 96-well plates at a ratio of 1:1. Supernatants were collected after 24 h and assayed for IFNγ by ELISA using DuoSet ELISA kit (R&D Systems) and LDH release  according to the manufacturer’s instructions.Human melanoma 624 wt cells were plated and treated with Tg, Nel, and Lop for 16 h. Then, the drugs were washed and 10Total RNA was isolated using TRI-reagent (Bio-Rad). Total RNA (1 μg) was reverse transcribed with an iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer’s instructions. Quantitative PCR was used to measure mRNA expression as follows: cDNA was mixed with 0.2 μM of both the forward and reverse primers in a final volume of 5 μl and mixed with 5 μl of iTaq universal SYBR Green Supermix (Bio-Rad). hRPLP0 was used as endogenous reference gene for PCR quantification. PCR was performed on CFX Connect™ Real-Time PCR Detection System (Bio-Rad). For polysome profiling, the combined phases were treated with 8 M guanidine hydrochloride and 1 mL of 100% cold ethanol, then incubated in − 20 °C overnight. The samples were spanned down at 20,000 g for 30 min at 4 °C, washed with 75% cold ethanol, and resuspended with 1 ml Trizol; then, RNA was extracted as mentioned above. The following primers were used: RPLP0 FW: 5′-CCAACTACTTCCTTAAGATCATCCAACTA-′3, REV: 5′-ACATGCGGATCTGCTGCA-′3; B7H6 FW: 5′-TCACCAAGAGGCATTCCGAC-′3, REV: 5′-TGGGGAAGCCACAACTTCAA-′3. ATF4 FW: 5′-ATGACCGAAATGAGCTTCCTG-′3, REV: 5′-GCTGGAGAACCCATGAGGT-′3. Primers’ quantitative efficiency was validated using standard curves.Cells were plated in equal densities, whenever needed. They were treated with 0.125 μg/ml of thapsigargin, 10 μM nelfinavir, or 20 μM lopinavir for the indicated time. Cells were then lysed using RIPA buffer  and analyzed by SDS-PAGE. Quantification of blots was performed with the Image Lab software. The following primary antibodies were used: B7H6 , ATF4 , Flag , p-eIF2α , total eIF2α , β-actin , α-tubulin , p97 . HRP-conjugated secondary antibodies (Goat anti-rabbit and Rabbit anti-mouse) were purchased from Jackson ImmunoResearch.A total of 624 wt cells were transfected using TransIT®-2020 (Mirus) reagent with Flag-ATF4 vector or transduced with Flag-B7H6 lentiviral vector. Forty-eight hours post transfection, cells were harvested and tested for Flag, ATF4, and B7H6 expression by immunoblotting.g, 30 min, 30 °C). The infected cells were incubated under normal conditions  for 48 h post-infection, then harvested and taken for analysis using flow cytometry. The presented analysis is gated only on the infected (GFP-expressing) cells.The virus used in HCMV infection experiments is an HCMV TB40/e_GFP mutant strain deleted for the genomic region encompassing US17-20. The Virus was generated and grown as previously described . For theU test or Kruskal–Wallis one-way analysis of variance to determine statistical significance at *p < 0.05.We applied a non-parametric Mann–Whitney The UPR through the IRE1 arm suppresses the transcription of MICA and MICB . To charDetection of B7H6 by immunoblotting is problematic. The commercial antibody used to detect B7H6 by flow cytometry does not recognize the protein in Western blotting applications, and the few antibodies used for B7H6 immunoblotting suffer from non-specific interactions. We were therefore concerned that the increase in B7H6 surface staining may be a technical artifact. For this purpose and for later experiments, as detailed below, B7H6 KO cells were prepared. Single-cell clones were screened with the best antibody we could obtain for Western. This effort identified a KO clone, for which the lack of surface B7H6 was validated by the flow cytometry antibody Fig. SC. This rTo further confirm the role of the PERK pathway in B7H6 regulation, we interfered with its signaling cascade with two drugs, each targeting the pathway at a distinct step. GSK2606414, directly inhibits PERK . Both inTo examine whether physiological ER stress also results in B7H6 induction, we infected the melanoma cells with human cytomegalovirus (HCMV), a virus that causes ER stress upon infection . CuriousPhosphorylation of eIF2α induces the expression of proteins, whose mRNA contains a non-productive upstream open reading frame (uORF) . An examWe decided to append the 5′UTR of B7H6 on the ORF of GFP, forming a translation reporter, termed 5′BG. As a control, we mutated the AUG codon of the uORF, termed AAG5′BG Fig. . The vecWe, therefore, assumed that the induction of B7H6 expression is due to increase in its mRNA levels. Indeed, the mRNA levels of B7H6 were elevated in wt cells by two-fold following Tg treatment. Total mRNA levels in PERK KO cells were significantly lower than in wt cells and were slightly affected by the treatment Fig. . This waThe main transcription factor of the PERK pathway is ATF4, induced by combined transcription and translation . ATF4 biThe induction of B7H6 by ER stress raises the intriguing possibility to promote B7H6 expression by drugs. The HIV protease inhibitors nelfinavir (Nel) and lopinavir (Lop) increase eIF2α phosphorylation by engaging different mechanisms , 33. TheWe also tested the effects of the drugs on PERK KO cells. As noted above, the basal level of B7H6 in PERK KO cells is lower. In distinction to Tg, both drugs were effective also in the PERK KO cells Fig. , suggestWe performed a time course analysis followed by immunoblotting and flow cytometry analyses for B7H6 to characterize the kinetics of B7H6 induction by the different pharmacological agents. B7H6 protein levels started to rise after 6 h of treatment Fig.  and accuNelfinavir also shows anti-cancer properties , 36 in aImmunotherapy has revolutionized traditional cancer treatment. Recent advances in using checkpoint inhibitors and adoptive cell therapy as well as clinical success have placed it at the center of attention for therapy development. In this context, melanoma shows a great albeit limited potential, with only 30% of patients responding to the treatment . UnderstAll three arms of the UPR are involved in cancer biology. The PERK pathway in particular is involved in almost all of the steps of cancer biology from initiation to therapy. For instance, PERK is activated and is required for Myc-dependent transformation  and the The PERK arm is responsible for global protein translation inhibition under ER stress. Some cellular proteins, such as ATF4, CHOP, and Bip, escape this inhibition and are induced when PERK is activated –44. The B7H6 mRNA was induced by ER stress. However, the magnitude of surface upregulation consistently exceeded the induction in transcript levels Fig. , suggestThe induction of B7H6 via Tg and under HCMV infection Figs.  and 2 leAn additional advantage to combine nelfinavir with B7H6-directed immunotherapy is the anti-cancer effects of nelfinavir as a standalone therapy  or in coESM 1(PDF 1350 kb)


Five samples contained vitamin D2: raw wattleseed (Acacia victoriae) (0.03 µg/100 g dry weight (DW)); fresh and dried lemon myrtle (Backhousia citriodora) leaves ; and dried leaves and berries of Tasmanian mountain pepper (Tasmannia lanceolata) . Fresh kombu (Lessonia corrugata) contained vitamin D3 (0.01 µg/100 g DW). Detected amounts were low; however, it is possible that exposure to ultraviolet radiation may increase the vitamin D content of plants and algae if vitamin D precursors are present.Vitamin D has previously been quantified in some plants and algae, particularly in leaves of the Solanaceae family. We measured the vitamin D content of Australian native food plants and Australian-grown edible seaweed. Using liquid chromatography with triple quadrupole mass spectrometry, 13 samples  were analyzed in duplicate for vitamin D ExtractThe samples were homogenized with 1 g ascorbic acid, 10 mL deionized water, 30 mL absolute ethanol, 2 g potassium hydroxide pellets, and 100 µL of 100 ng/mL deuterated internal standard mix and made to 50 mL with deionized water. The headspace was flushed with nitrogen gas, capped, and placed in a shaker for saponification overnight. The samples underwent centrifugation and 10 mL of the ethanol layer was extracted onto diatomaceous earth Solid Phase Extraction (SPE) cartridges (ChemElute Agilent). The organic soluble compounds were washed off with two 30 mL aliquots of petroleum spirits. The washes were collected into 80 mL glass EPA vials and then evaporated to dryness under high purity nitrogen gas. The residue was reconstituted into 400 µL heptane and transferred to a LC vial containing a 400 µL glass insert. The prepared extracts were stored at −20 °C.The reagent, PTAD  reacts non-specifically with dienes under a reaction mechanism called the Diels–Alder Reaction. While in theory, excess PTAD is added to derivatize the vitamin D analogues, samples with high diene content may limit the amount of PTAD available for derivatization. Therefore, where samples were determined to have high diene content, extract clean-up via normal phase chromatography fraction collection was performed. The extracts were inspected for cold precipitate: if present, the liquid extract was transferred to a fresh 400 µL glass insert. Of the remaining liquid extract, 200 µL were injected onto a normal phase chromatographic system with a silica column, 1 mL/min 2% isopropyl alcohol in heptane mobile phase, and a photodiode array detector set to 265 nm. Vitamin D and 25(OH)D fractions were collected.Fractions of vitamin D and 25(OH)D were combined and evaporated under high purity nitrogen gas. The dry material was reconstituted in 200 µL of dry acetonitrile containing 1 mg/mL of 4-phenyl-1,2,4-triazole-3,5-dione (PTAD) and transferred to a fresh LC vial. Two hours were allowed to complete derivatization. The sample was evaporated under high purity nitrogen gas. The dry material was reconstituted in 100 µL of methanol and water (70:30), transferred to a fresh 400 µL glass insert, and placed into an LC vial. A limit of quantitation was conservatively set at 0.05 µg/100g, which is half of the ‘spiked’ recovery level of 0.1 µg/100g.3, 86–104%; vitamin D2, 76–105%; 25(OH)D3, 85–114%; and 25(OH)D2, 90–114%. The recovery for 25-hydroxyvitamin D3 in wattleseed (roasted/milled/ground seed) could not be determined due to a matrix interference.The recoveries were determined for each sample analyzed. The recoveries at the 0.1 µg/100 g ‘spiked’ recovery level were as follows: vitamin D2, vitamin D3, 25(OH)D2, and 25(OH)D3 using LC-QQQ . The calibration samples of vitamin D2, vitamin D3, 25(OH)D2, and 25(OH)D3 were prepared. The calibration concentrations (in ng/mL) were 0, 2.5, 5, 7.5, 10, 15, 25, 50, 75, and 100. Each calibration sample also contained 10 ng/mL of deuterated internal standard for each vitamer  tested. The calibrations and samples were analyzed using 1290 Infinity Series LC System/6460 Triple Quad liquid chromatography–tandem mass spectrometry  fitted with a Jet Stream ESI source in positive ion mode using a Supelco Ascentis Express C18 10 cm × 2.1 mm, 2.7 µm LC chromatographic column .The samples were analyzed for vitamin DFor each vitamer analyzed, each sample was tested in duplicate, and duplicate values were averaged to obtain one mean value for each sample. A third sample, spiked with the same vitamer, was analyzed for each sample tested to provide quality control data. The mean percentage recovery and mean relative percentage difference were calculated for each vitamer. At the time of writing, the expected limit of detection, post validation study, is expected to be 0.05 µg/100 g .2, vitamin D3, 25(OH)D2, and 25(OH)D3 was 96%, 98%, 101%, and 94%, respectively. Across all samples, the mean relative percentage difference between duplicate samples for vitamin D2, vitamin D3, 25(OH)D2, and 25(OH)D3 was 71%, 15%, 50%, and 56%, respectively.The mean recovery percentage across all samples for vitamin D2 was found in dried lemon myrtle leaves (0.24 µg/100 g) and the dried leaves and berries of Tasmanian mountain pepper . In addition, three samples contained detectable vitamin D metabolites at levels below the limit of quantitation. Approximate levels are provided for indicative purposes only. Vitamin D2 was detected in raw wattle seed (≈0.03 µg/100 g) and fresh lemon myrtle leaves (≈0.03 µg/100 g). Vitamin D3 was detected in fresh kombu (≈0.01 µg/100 g). There were no detectable vitamin D metabolites in the other samples  and hops (Humulus lupulus L.) [3 and, in some cases, 25(OH)D3 have previously been detected in the leaves of tomato [We detected low levels of vitamin Dmination . Vitaminulus L.) ,44. Vitaf tomato ,25,26, wf tomato ,25, potaf tomato , day blof tomato , zucchinf tomato , and alff tomato ; howeverDacrydium cupressinum), a podocarp native to New Zealand, was found to contain substantial amounts of both vitamin D2 and D3 [2 and D3 contents of rimu berries were reported as 70 µg/100g and 11.5 µg/100g, respectively, although no quality control data were provided. In another study, the precursors of vitamin D2 and D3  were detected in plant oils [3 and 25-hydroxyvitamin D3 content of leaves and cell cultures of certain plants increases after ultraviolet (UV) irradiation [3 content of tomato (Solanum lycopersicum L.) leaves by almost 60 times to 100 ng/g, compared with 1.7 ng/g in non-UV-exposed leaves [Recently, the fruit of the rimu tree  in much larger quantities than were found in our study [3 in kombu (Lessonia corrugata) measured at 0.01 μg/100 g. In other studies, Japanese wireweed (Sargassum muticum) was found to contain 90 μg/100 g, while vitamin D2 and D3 contents in microalgae ranged from not detected to 3900 μg/100 g and 2.2–271.7 μg/100 g, respectively [2 and D3 content than those harvested in autumn and winter [3 in kombu and no vitamin D2 or vitamin D3 in wakame, the algae tested in our study were harvested in the winter months and were not sundried or exposed to UV radiation after harvest.croalgae , was fircroalgae . Since tur study ,31,32. Vectively ,31,32. Eectively ,48. The d winter . Althoug2, vitamin D3, 25(OH)D2, and 25(OH)D3, due in part to the presence of interfering compounds such as chlorophyll and lipophilic pigments [Plant and algal matrices present challenges for the quantification of vitamin Dpigments . Therefopigments . When copigments . To our pigments ,48, we aIn conclusion, this study has demonstrated the high sensitivity of LC-QQQ methodology, which will be used in future studies in search of natural dietary sources of vitamin D. Our results show that the selected Australian native plants and algae have very low levels of vitamin D. However, given that the vitamin D precursors, ergosterol and 7-dehydrocholesterol, have previously been found in both plants and algae, testing the effect of exposure to UV radiation on the vitamin D content of Australian native food plants and Australian-grown edible seaweed is warranted. Also, a larger sample size across a greater number of plant species and habitats will increase the likelihood of identifying nutritionally relevant amounts of vitamin D. An important factor in future studies will be taking the seasonal effect of vitamin D into account in natural ecosystems, because this will relate back to the levels of solar UV radiation for potentially increasing vitamin D levels.


The Scan Electron Microscope Images (SEM), X-ray Diffraction and Fourier Transform Infrared Spectroscopy (FTIR) dataset has been outlined investigating morphological features change of native sugarcane bagasse, as an agro-industrial lignocellulosic feedstock waste and a potential for cellulose biopolymer extraction, pretreated by alkali (sodium hydroxide) followed by an acid step (sodium bisulfate) in an exothermic in-situ one step, pretreated by acid (sulfuric acid) followed by residual solid fraction alkali pretreatment (sodium hydroxide) in a two separate individual steps and finally after the enzymatic cellulolysis. Data explained herein helps to extend and add to knowledge regarding the impact unlikeness of two different pretreatment methodologies utilize the same chemicals and relatively same concentrations on the cellulosic fiber morphological features and consequently its enzymatic accessibility. This data are related to Egypt Patent Office application, 1349/2017, entitled “In-situ sodium hydroxide-sodium bisulfate sugarcane bagasse pretreatment for biofuel production”,Zohri et al., 2017 [1]. Chipped into small pieces 1 cm length, washed several times with water and stored in refrigerator prior to the pretreatment process.2.2Pretreatment was carried out in autoclave, Systec VE-75. using the procedures described In-situ sodium hydroxide-sodium bisulfate 2.2.18 gm sugarcane bagasse, 1–2 cm length, immersed in 80 ml sodium hydroxide 1.3% and autoclaved at 120 °C for 40 min. Then a 20 ml water containing 1.45 ml sulfuric acid 98% is added while the slurry temperature 80 °C and the temperature kept at 105 °C for further 40 min. The resulting solid fraction was washed with water and post treated by 0.2% sodium hydroxide dissolution in cold for lignin recovery and cellulose fiber separation, solid yield 47.5%.2.2.210 gm sugarcane bagasse, 1–2 cm in length, immersed in 100 ml sulfuric acid 1% and autoclaved at 120 °C for 40 min. The resulting solid fraction was filtered and washed with a plenty of water and dried.Dry residual solid fraction was immersed in sodium hydroxide solution 2%, 1:10 solid to liquid ratio and autoclaved at 120 °C for 40 min, solid yield 28.3%.2.3Bagasse morphology was analyzed before and after pretreatment in addition to after enzymatic hydrolysis. Samples were dried and coated with gold and were imaged using scan electron microscopy, JOEL-JSM 5400 LV (Japan).2.4X-ray diffraction data were obtained in Philips PW 1710 using monochromatic CuKα radiation (1.54 Å), 40 KV and 30 mA setting. The crystallinity index for all the samples was calculated according to the procedure proposed in 2.5FTIR spectra were recorded on Nicollet 6700 spectrophotometer using potassium bromide.2.6Enzymatic hydrolysis of pretreated sugarcane bagasse was carried out at a substrate ratio of 2.5% (wt/v) in 0.05 M sodium citrate buffer (pH 5.0), at 45 °C. The cellulase applied consist of 12 filter paper units of Cellic Ctech2, 0.12 ml, per 1 g solid substrate . The hydrolysis yield expressed in glucose yield was determined as described by Maeda et al. Fermentation was carried out using single batch SSF, Separate Hydrolysis and Fermentation, utilizes fed-batch enzymatic hydrolysis at 15% water solid load, centrifuging then collecting the clear sugar solution nutrient enriched with 5 g/l peptone, 3 g/l yeast extract, 3 g/l malt extract and 1 g/l Saccharomyces cerevisiae affording 78.2% fermentation efficiency.The following


The suppressor of cytokine signaling 2 (SOCS2) acts as substrate recognition subunit of a Cullin5 E3 ubiquitin ligase complex. SOCS2 binds to phosphotyrosine-modified epitopes as degrons for ubiquitination and proteasomal degradation, yet the molecular basis of substrate recognition has remained elusive. Here, we report co-crystal structures of SOCS2-ElonginB-ElonginC in complex with phosphorylated peptides from substrates growth hormone receptor (GHR-pY595) and erythropoietin receptor (EpoR-pY426) at 1.98 Å and 2.69 Å, respectively. Both peptides bind in an extended conformation recapitulating the canonical SH2 domain-pY pose, but capture different conformations of the EF loop via specific hydrophobic interactions. The flexible BG loop is fully defined in the electron density, and does not contact the substrate degron directly. Cancer-associated SNPs located around the pY pocket weaken substrate-binding affinity in biophysical assays. Our findings reveal insights into substrate recognition and specificity by SOCS2, and provide a blueprint for small molecule ligand design. The suppressor of cytokine signaling 2 (SOCS2) is a component of the Cullin5 E3 ubiquitin ligase complex. Here the authors provide insights into substrate recognition and specificity of SOCS2 by determining the crystal structures of the SOCS2-ElonginB-ElonginC in complex with phosphorylated peptides from two of its substrates the growth hormone receptor and erythropoietin receptor. The Janus kinase (JAK)—signal transducer and activator of transcription (STAT) signaling pathway plays a critical role enabling cells to respond to specific cytokines by regulating gene expression. Suppressor of cytokine signaling (SOCS) proteins, which comprise of cytokine inducible SH2-containing protein (CIS) and SOCS1–SOCS7, negatively regulate cytokine receptors and inhibit the JAK-STAT signaling pathway2.Cytokines are small glycoproteins that play important roles in the differentiation, development and function of lymphoid and myeloid cells5, followed by a central Src-homology 2 (SH2) domain that recognizes a phosphotyrosine (pY) containing sequence6, and a C-terminal SOCS box that interacts with the adapter ElonginB-ElonginC complex (EloBC)9. All SOCS proteins bind to EloBC and recruit Cullin5 with high specificity, forming different SOCS-EloBC-Cullin5-Rbx2 (CRL5SOCS) E3 ligases that catalyze ubiquitin transfer and subsequent proteasomal degradation of specific substrates, as a mechanism to regulate diverse biological processes13. SOCS proteins serve as substrate recognition modules that impart substrate specificity to each CRL5SOCS E3 complex.SOCS proteins share a conserved domain architecture comprising of an N-terminal extended SH2 subdomain (ESS) that functions as a bridge at the interface between the SH2 domain and the SOCS box enabling ubiquitination of captured substrate14; (2) Blocking STAT activation by competing for receptor pY sites15; (3) Targeting the receptor for proteasomal degradation via SOCS E3 ligase activity17. Some of the SOCS-substrate interactions have been structurally characterized, including SOCS1-JAK18, SOCS3-gp13019, SOCS3-gp130-JAK220, and SOCS6-cKit21.Expression of SOCS proteins is induced by cytokine stimulation. Upon cytokine binding, the oligomerized receptors activate the JAK family kinases that phosphorylate specific tyrosine residues on the receptor, including the docking sites for the STAT proteins. The docked STAT proteins are sequentially phosphorylated, they dimerize and translocate into the nucleus, initiating gene transcription of several downstream proteins including the SOCS proteins. SOCS proteins suppress the JAK-STAT pathway via three distinct but often concomitant mechanisms: (1) KIR mediated direct JAK inhibition25. SOCS2 has been shown as the primary suppressor of growth hormone (GH) pathway where a gigantism phenotype was observed in a SOCS2−/−mice26. Paradoxically, the SOCS2 overexpressed transgenic mice also led to the same phenotype27. Attenuation of GHR signaling relies on two phosphorylation sites at GHR that are recognized by SOCS228. The pY487 site of GHR interacts with CRL5SOCS2 E3 ligase that targets the GHR for ubiquitination and proteasomal degradation16. A downstream pY595 site interacts with SOCS2, STAT5b and SHP2 (SH2 domain-containing phosphatase 2), enabling SOCS2 to inhibit the signaling by blocking this receptor site from STAT5b30. Nonetheless, deletion of both sites is required to remove the inhibitory effect of SOCS2 on the GH signaling28. Analysis of the binding affinity of SOCS2 for these two phosphorylation sites of GHR reveals that the pY595 region exhibits a higher affinity towards SOCS2 (KD = 1.6 μM) compared to the pY487 region (KD = 11.3 μM)32. An 11-mer phosphorylated peptide spanning the pY595 region of GHR was sufficient to pull down the whole CRL5SOCS2 complex from human cell lysates31 as well as CIS, the closest homolog to SOCS2 from the same family, which plays a role in anti-tumor immunity controlling the differentiation of CD4 T helper cell, and the IL-2 and IL-4 response33. In addition to GHR, other substrates have been identified to interact with SOCS2, including the erythropoietin receptor (EpoR) at pY42634, the leptin receptor at pY107735, the epidermal growth factor receptor36 and the insulin-like growth factor-I receptor37. The first crystal structure of SOCS2-ElonginB-ElonginC (SBC) was reported in 200632, however the structural basis for substrate recognition by SOCS2 has yet remained elusive.SOCS2, one of the members of the SOCS family, is implicated in disorders of the immune system, central nervous system and cancer, and is thus emerging as a promising therapeutic target Here, we determine the co-crystal structures of SBC in complex with phosphorylated epitope peptides from its physiological targets GHR and EpoR. Our structures reveal the peptides are accommodated in an extended conformation to capture specific interactions with SOCS2. A key flexible region of SOCS2, known as the BG loop, is defined in the electron density and shown not to contact the bound substrates. Structural analyses supported by biophysical and mutagenesis investigations identify hotspot residues on the substrate degrons and functionally elucidate disease-relevant single nucleotide polymorphisms (SNPs) of SOCS2. Our findings reveal fresh insights into the molecular recognition and selectivity between SOCS2 and target substrates, and provide an important template for future structure-guided ligand design.KD of 6.9 μM) and GHR_pY595 (KD of 1.1 μM) was measured by isothermal titration calorimetry (ITC), and found to be consistent with the literature32  eventually yielded high-resolution datasets.To elucidate the molecular basis of substrate recognition by SOCS2, we subjected the SOCS2-ElonginB-ElonginC (SBC) complex to extensive co-crystallization trials with 11-residue phosphopeptides of either EpoR or GHR that span the regions surrounding Tyr426 and Tyr 595 region, respectively. The affinity of SBC for EpoR_pY426  was solved and refined at 2.69 Å with 19.64%  40 Fig. . Electro 40 Fig. . A class 40 Fig. . Additio 40 Fig. . Multipl 40 Fig. .Table 1CRwork and 22.66% Rfree . The two peptides run in an anti-parallel direction relative to each other across the SH2 domain, with well-defined electron density surrounding them both  and solved the structure at 1.98 Å resolution with 19.00% oth Fig. . One of oth Fig. . In contoth Fig. .Fig. 2StThe unusual simultaneous binding observed for the GHR substrate peptide to SOCS2 SH2 domain is imparted mainly by the region comprising Ser(+2) to Val(+6) from each peptide, which pair such that they form an anti-parallel beta sheet Fig. . ExtensiD of 2.3 μM by ITC, Supplementary Fig. 2), still two copies of the GHR_pY595 peptide, but no GHR_pY487, are observed bound, yielding a structure very similar to the previous SBC-GHR structure  to Arg186 (SOCS box) and Pro140 (BG loop) to Ile90 (EloB), as clearly defined by the unbiased omit map at this region  and BG loop. In the absence of substrate peptide, the EF loop curls up placing the Ile110 and Cys111 at the hydrophobic SH2 domain  was monitored as a mean to quantify the extent of the competition between the tested peptides and the spy molecule. A Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence was applied to estimate the spin–spin relaxation time (T2) of the spy molecule in the absence and presence of protein45. By adding competitor to disrupt the protein-spy interaction, the binding affinity of a competitor can be calculated based on the degree of displacement of the spy47. The spy molecule used in our assay is compound 3, a fluorinated pY analog that specifically binds to the pY pocket with a KD of 50 μM  and Ki (NMR) values correlated well (R2 of 0.74)  and First, we focused on the unique interaction formed by Val(−3) of GHR_pY595, which inserts into a small hydrophobic cavity of SOCS2 Fig. . This in31. The resulting library comprised of the original wild-type sequences, ten derivatives from GHR_pY595 and nine from EpoR_pY426, and was characterized in parallel using SPR and 19F NMR competition assay (Table D) compared to the wild-type. In contrast, a similar (at least two-fold) weakening in binding affinity was observed in the EpoR_pY426 peptide upon alanine substitution at pY(−1), pY(+2), and pY(+3). These results are consistent with observations from our crystal structures that peptide-SOCS2 binding is mediated by hydrophobic interaction including pY(−3), pY(+3), and pY(+5) on the GHR_pY595 and pY(+2) and pY(+3) on the EpoR_pY426. The binding affinity for each peptide also dropped by at least two-fold with the Thr/Ala substitution at pY(−1) position, indicating the importance of the residue just upstream of pY.Next, to map the relative importance and contribution of each individual amino acids to the binding affinity with SBC, alanine scan of the substrate peptides was invoked. The peptide sequences were designed such that individual amino acids were separately mutated into alanine except pY, which is known to abolish binding if mutated even to unphosphorylated Y48. We thus next decided to characterize the interaction of selected SNP SOCS2 mutants with substrate peptides GHR_pY595, EpoR_pY426 and GHR_pY487 by SPR  on SOCS2 are reported in the Catalog of Somatic Mutation in Cancer database (COSMIC) as potentially linked to cancers such as tumors of the lung, breast, and pancreasPR Table . InspectPR Table . All mutKD values could not be measured with these protein mutants. For the R96Q mutation no signal response was detected, suggesting that binding was completely abolished and highlighting the most disruptive of the mutations studied herein. Our result is consistent with evidence by Rupp et al. that the point mutation R96C abrogates substrate binding by SOCS249.The L106V and C133Y mutations did not affect binding affinities compared to wild-type (Table R2) of spy molecule 3 using 19F NMR spectroscopy , indicating that these mutations retain pY recognition. In contrast, mutations on N94 and R96 led to R2 values for spy molecule 3 comparable to those in the absence of protein (around 3–4 s−1), consistent with a loss of pY binding. Together, the 19F NMR and the SPR data consistently elucidate abrogated substrate binding for SOCS2 SNP mutations N94D, R96L, R96Q, which are located around the pY pocket.In addition to SPR, we evaluated to what extent SNP mutants retain competence to bind substrate pY by monitoring the transverse relaxation rate , pY(+1) and pY (+3) positions of the GHR_pY595 and EpoR_pY426 peptide contains similar properties; whereas residues at pY(−3), pY(+2), and pY(+5) position are different in properties and sizes Fig. . In part21. In the SOCS3 and SOCS6 peptide-bound structures, the BG loop folds up as a hairpin interacting with the substrate peptide, forming a triple-stranded β sheet structure , suggesting that this region does not participate in substrate recognition. Nevertheless, interestingly, a similar triple-stranded β sheet structure is observed in the SBC-GHR2 structure, where the peptide B replaces the first β-sheet of the BG loop and makes backbone interactions with the BG loop and peptide A . Zhang et al. suggested that the dimerization of peptide binding in SHP-2 requires at least one pY containing peptide and leads to enhanced binding affinity. In the case of the SBC-GHR complex, despite preparing several protein–peptide samples for co-crystallization at 1:1 molar ratio, all dataset collected from crystals were consistent with a 1:2 (protein–peptide) binding mode. In both cases, the binding of the non-canonical binding peptide relies on hydrophobic interaction with the SH2 domain hydrophobic core, and backbone to backbone interaction with the canonical binding peptide and protein  for recognition Fig. . Howeverane Fig. . SOCS2 m57. Breast, lung, liver, and ovarian cancer have been correlated with downregulation in SOCS263. In addition to the JAK-STAT pathway, a recent study has identified the involvement of SOCS2 in the NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) pathway that regulates the immune and inflammatory responses65. NF-κB is found to be constitutively activated in many types of cancer and influences a diverse array of pro-tumorigenic functions, therefore NK-κB plays a pivotal role in cancer initiation and progression66. SOCS2 negatively regulates TNFα induced NF-κB activation by targeting NDR1, a serine-threonine kinase, for proteasomal degradation. Hence SOCS2 deficiency may lead to increased levels of NDR1, which results in aggressive behavior of PC3 prostate cancer cells64. These evidences highlight the potential in targeting SOCS2 for drug discovery for inflammation and cancer biology. We have revealed structural insights into the SOCS2-peptide interactions by X-ray crystallography and identified hotspot using alanine scanning, mutation study and SNPs study. This information provides a template to guide the structure-based rational design of SOCS2 ligands that are instrumental in the development of novel chemical tools to probe SOCS2 biology, and in the quest for novel small molecule ligands binding to SOCS2 as potential therapeutics. SOCS2 binders at the pY binding pocket can be used as inhibitors of the CRL5SOCS2, which would be expected to prevent degradation of target substrate receptors, thus prolonging the activity of cytokine signaling pathway and upregulating expression of endogenous STAT5b-responsive gene expression. In a distinct application, a SOCS2 binder could provide a novel E3 ligase ligand for designing new chemical degraders to hijack SOCS2 CRL activity and trigger the degradation of unwanted proteins inside cell69. This approach, also known as proteolysis targeting chimeras (PROTACs), offers the advantage of inducing rapid and selective intracellular depletion of the target protein, as opposed to mere blockade of a single interaction or activity, which pairs more closely to genetic target validation and often results in greater maximal efficacy of intervention in a signaling pathway. PROTAC-mediated protein degradation has been shown to occur at very low compound concentration (pM to nM range), well below the range of inhibitory concentrations for the hijacked E3 ligase71. PROTACs potentially allows targeting of intractable protein targets that are beyond the reach of conventional small-molecule approaches that require full occupancy of a target binding site e.g., receptor antagonists and enzyme inhibitors. A limited set of E3 ligases have been targeted so far for PROTACs, notably VHL71 and cereblon73, so extending the approach to other ligandable E3 ligases would be an important advance to the field. The usage of SOCS2 binders for PROTACs may be specific to SOCS2 expressing cells, thus could provide an additional layer of tissue specific degradation of target proteins. For example, PROTACs designed using SOCS2 binders as recruiters would be ideal for degrading targets specifically in cells affected with leukemia and gastrointestinal sarcoma which have been reported to have upregulated SOCS2 expression74. Low levels of SOCS2 in other normal tissues would help minimize toxicity. On the other hand, a pan-SOCS recruiting PROTACs would help diversifying the target tissue range. Our peptide-bound co-crystal structures suggest that SOCS2 might be ligandable and provide a blueprint for the rational structure-guided design of novel SOCS2 inhibitors and SOCS2 ligands for PROTACs.SOCS2 is an attractive therapeutic target due to its links to cancer, diabetes, neurological and inflammatory diseases39. Briefly, SBC was co-expressed in E. coli BL21(DE3) from pLIC (His6-SOCS2) and pCDF (EloBC) plasmids. Protein expression was induced with isopropyl β-d-1-thiogalactopyranoside at 18 °C for 12 h. After cell lysis, SBC protein was found in the soluble fraction and purified by affinity chromatography using a HisTrap column . Following tag cleavage with tobacco etch virus (TEV) protease and a second HisTrap column the desired untagged protein eluted in the flow-through fractions. SBC was finally purified by size-exclusion chromatography on a Superdex 75 16/600 column  in 25 mM HEPES, pH 7.5, 250 mM NaCl and 10 mM DTT. SOCS2 mutants N94D, R96L, R96Q, L106V or C133Y were introduced using PCR-based site-directed mutagenesis (details of the primers used are in Supplementary Table The human SOCS2 (amino acids 32–198) and the ElonginB (amino acids 1–104) and ElonginC (amino acids 17–112) plasmids were used for protein expression as previously reported38. SER-assisted crystallization attempts yielded crystals with the K115A/K117A/Q118A SOCS2-EloBC (SKKQBC). Five times molar excess of GHR_pY595  was incubated with SKKQBC, followed by removing unbound peptide using a protein concentrator. Sample was concentrated to 22 mg ml−1 with an additional 0.1 M of sodium cacodylate pH7.2 added to the sample. Diffraction-quality crystals were obtained with 0.005 M Cobalt (ll) chloride, 0.1 M MES pH 6.5, 1.0 M ammonium sulfate at 4 °C using hanging drop vapor diffusion method at 2:1 protein:precipitant ratio. Crystals were cryo-protected using 20% MPD prior to vitrification in liquid nitrogen.To improve crystallization, surface entropy reducing mutations were introduced into SOCS2 construct (amino acids 32–198). Three mutation clusters  were identified with the SER server75 and scaling and merging with AIMLESS within the CCP4 program suite77. The experimental phases were obtained by identifying the positions of arsenic atoms on the surface of SBC39, using MR-SAD phases in the PHENIX software suite79. The structure was reconstructed by AutoBuild81 and manually built in Coot82. The resulting structure was refined iteratively with REFMAC583.Diffraction data were collected at 100 K at Diamond Light Source beamline i04 using Pilatus 6M-F detector at 0.98 Å wavelength. Indexing and integration was processed by XDSKKQBC (5 mg ml−1). Unbound peptide was removed by a protein concentrator (sartorius Vivaspin) while the mixture was concentrated to 20 mg ml−1 concentration. Sodium cacodylate pH7.2 was added to a final concentration of 0.1 M prior to crystallization. Crystallization drops were set up in a ratio of 1:1 protein:precipitant in 18% ethanol, 0.1 M HEPES pH7.5, 0.1 M MgCl2 using hanging drop at 4 °C. Crystals were cryo-protected using 20 % PEG400 prior to flash-cooled.Five times molar excess of EpoR_pY426 (ASFEpYTILDPS-amide) was incubated with S75 and AIMLESS77. The phase was obtained by molecular replacement (MR) using Phaser79 with the coordinates of SOCS2-EloB-EloC (PDB ID: 2C9W) as a search model. The presence of the EpoR_pY426 was observed in the initial electron density map. Model building was conducted manually with Coot82 and refined with cycles of retrained refinement with REFMAC583.Diffraction data were collected at 100 K on beamline i24 at Diamond Light Source. Data were recorded to Pilatus3 6M-F detector at 0.97 Å wavelength. Data were indexed, integrated, and reduced using XDSKKQBC at 1:1:1 stoichiometric ratio with a final concentration of 20 mg ml−1 and additional 0.1 M sodium cacodylate pH 7.2. Drops of the complex were mixed 2:1 with 0.005 M cobalt chloride, 0.1 M MES pH6.5 and 1.0 M ammonium sulfate in the sitting-drop vapor diffusion format at 4 °C. 20% MPD was applied to crystal before flash-cooling.GHR_pY595 (PVPDpYTSIHIV-amide) and GHR_pY487 (NIDFpYAQVSDI-amide) were mixed with S2 co-crystal was at 100 K on beamline i24 at Diamond Light Source. Images were indexed, intergraded, and reduced using XDS75 and AIMLESS77. A molecular replacement solution was obtained by Phaser79 using SBC-GHR as search model. Refinement was performed using REFMAC583 and model building was performed in COOT82.Data collection of the SBC-GHR13C spectra were 1H decoupled. Chemical shifts (δ) are reported in ppm relative to solvent  as internal standard. High Resolution Mass Spectra (HRMS) were recorded on a Bruker microTOF. Low resolution MS and analytical HPLC traces (LC-MS) were recorded on an Agilent Technologies 1200 series HPLC connected to an Agilent Technologies 6130 quadrupole LC/MS, connected to an Agilent diode array detector. The column used was a Waters XBridge column  and the compounds were eluted with a gradient of 5−95% acetonitrile/water +0.1% formic acid over 3 min. Preparative HPLC was performed on a Gilson Preparative HPLC System with a Waters X-Bridge C18 column  and a gradient of 5% to 95% acetonitrile in water over 10 min, flow 25 ml min−1, with 0.1% formic acid in the aqueous phase.All chemicals, unless otherwise stated were commercially available and used without further purification. Solvents were anhydrous and reactions preformed under positive pressure of nitrogen. Flash column chromatography was performed using a Teledyne Isco Combiflash Rf or Rf200i. As prepacked columns RediSep Rf Normal Phase Disposable Columns were used. NMR spectra were recorded on a Bruker 500 Ultrashield. O-bis(dimethylamino)phosphono)-l-tyrosine84  and NaHCO3  were dissolved in the mixture THF/H2O = 1:1 (10 ml) and N-(benzyloxycarbonyloxy)succinimide  was added. The reaction mixture was stirred at room temperature overnight. After the addition of 5% NaHSO4 the product was extracted with ethyl acetate, washed with brine, dried over MgSO4, and concentrated by rotary evaporation under reduced pressure. After drying, Cbz-O-bis(dimethylamino)phosphono)-l-tyrosine 1  was obtained as pale yellow. 1H NMR (CD3OD): 2.69 , 2.92 , 3.18 , 4.40 , 5.03 , 7.06 , 7.21 , 7.25–7.36 . 31P NMR (CD3OD): 18.2. LC-MS (m/z): [M+H]+ calcd. for C21H29N3O6P, 450.17; found, 450.2.1 , HATU , HOAt  and DIPEA  in DMF (1 ml), 2 M methylamine solution in THF (0.5 ml) was added under stirring at room temperature. After 2 h, LC-MS analysis showed complete conversion of the starting material and formation of the desired product. The mixture was diluted with ethyl acetate, washed with 5% NaHSO4, brine, dried over MgSO4, and concentrated by rotary evaporation under reduced pressure. The crude product was dissolved in the mixture ethanol/ethyl acetate = 1:1 (8 ml). Hydrogenation was carried out using H-Cube at 80 ˚C, Pd/C, 1 atm, at 1 ml min−1. The solvent was evaporated under vacuum to afford 2  which was directly used in the next step without any further purification. 1H NMR (CD3OD): 2.66 , 2.71 , 2.81 , 2.96 , 3.51 , 7.09 , 7.20 . 31P NMR (CD3OD): 18.3. LC-MS (m/z): [M+H]+ calcd. for C14H26N4O3P, 329.17; found, 329.2.To a mixture of the compound 2  and DIPEA  in DCM (2 ml) was cooled to −78 °C, and trifluoroacetic anhydride  was added. The reaction mixture was stirred 1 h at −78 °C. After solvent evaporation the residue was dissolved in acetonitrile (0.5 ml) and 2 M HCl was added (2 ml). The mixture was stirred at room temperature overnight until no presence of the starting materials was detected by LC-MS. The solvents were evaporated and residue was purified by HPLC to afford compound 3  as a white solid. 1H NMR (CD3OD): 2.69 , 2.96 , 3.15 , 4.57 , 7.13 , 7.22 . 13C NMR (CD3OD): 26.3, 37.8, 56.5, 117.3 , 121.3 , 131.3, 134.1, 151.9 , 158.7 , 172.4. 31P NMR (CD3OD): 3.7. 19F NMR (CD3OD): −75.6. HRMS (m/z): [M+H]+ calcd. for C12H15F3N2O6P, 371.0620; found, 371.0599.A solution of the compound −1) on an INTAVIS ResPepSL automated peptide synthesizer. O-(dibenzylphosphono)-N-Fmoc-l-tyrosine was synthesized as described below. The peptides were cleaved with 2.5% triisopropylsilane and 2.5% water in TFA. The crude peptides were isolated from the cleavage mixture by precipitation with cold ether, dissolved in the mixture water/DMF = 1/1 and purified by preparative HPLC under the following conditions: Waters X-Bridge C18 column , gradient of 5–95% acetonitrile in water over 10 min, flow 25 mL min−1, with 0.1% formic acid in the aqueous phase, UV detection at λobs = 190 and 210 nm. The poorly there soluble peptides were purified according to literature procedure85: the impurities were extracted by DCM from the solution of peptides in 20% acetic acid. The purity and identity of the peptides were determined by the analytical LCMS on an Agilent Technologies 1200 series HPLC connected to an Agilent Technologies 6130 quadrupole LC/MS linked to an Agilent diode array detector  and tert-butyldimethylsilyl chloride  were added. After 15 min 4,5-dicyanoimidazole  and diisopropylphosphoramidite  were added and the reaction mixture was stirred at room temperature for 4 h. After cooling to 0 °C 70% tert-butyl hydroperoxide  was introduced. After stirring for 2 h at 0 °C, 10% Na2S2O5 (20 ml) was added and stirring continued for one more hour. The product was extracted with ethyl acetate, washed with a 5% solution of KHSO4, brine, dried over MgSO4, concentrated by rotary evaporation under reduced pressure, and further purified by column chromatography on silica gel using a gradient elution of 0–10% of MeOH in DCM to afford O-(dibenzylphosphono)-N-Fmoc-l-tyrosine  as a pale yellow solid. NMR spectra were in agreement with the published data86.To a solution of Fmoc-tyrosine  in anhydrous THF (12 ml) H), and stoichiometry (N) using MicroCal ITC-ORIGIN Analysis Software 7.0 .Experiments were performed with ITC200 instrument  in 100 mM HEPES pH 7.5, 50 mM NaCl, 0.5 mM TCEP at 298 K stirring the sample at 750 rpm. The ITC titration consisted of 0.4 μl initial injection  followed by 19 of 2 μl injections at 120 s interval between injections. The GHR_pY595 peptide , EpoR_pY426  and GHR_pY487  were directly titrated into SBC (50 μM). Binding data was subtracted from a control titration where peptide was titrated into buffer, and fitted using a one-set-of-site binding model to obtain dissociation constants, binding enthalpy . All data were double-referenced for reference surface and blank injection. The processed sensograms were fit to a steady-state affinity using a 1:1 binding model for KD estimation  in 20 mM HEPES pH7.5, 150 mM NaCl, 1 mM TCEP, 0.005% Tween20 buffer at 10 °C. Biotinylated wild-type SBC and mutants were immobilized onto a chip surface and injected a series of seven concentrations  of peptide across the sensor surface for 60 s contact time and 120 sec dissociation time at 30 μl min1 = −35451 Hz (−75.3 ppm). Protein was used at 5 μM and spy molecule 3 was used at 100 μM in buffer containing 20 mM HEPES pH 8, 50 mM NaCl, 1 mM DTT, 20% D2O. The GHR related peptides including wild-type, V(−3)R, V(−3)Y and alanine scanning peptides were used at 10 μM, whereas the EpoR related peptide including wild-type and alanine scanning were used at 50 μM. All NMR data were processed and analyzed using TopSpin (Bruker).All NMR experiments were conducted using AV-500 MHz Bruker spectrometer equipped with a 5 mm CPQCI 1H/19F/13C/15N/D Z-GRD cryoprobe) at 298 K. Spectra were recorded using 80 scans of a CPMG pulse sequence that attenuates broad resonances. A CPMG  delay of 0.133 s was used, to maximize the difference between the signal intensity of spy molecule alone and in the presence of protein . The transmitter frequency was placed close to the resonance of OKi) was calculated by adapting the method described by Wang et al.47 Briefly, peptides’ Ki values were obtained from the equation below:I0] and [P0] are the total concentrations of the competitor (peptide inhibitor) and protein, respectively, used in the experiment, while [PI] and [PL] are the free concentrations of protein–peptide and protein–spy complexes, which are unknown.The dissociation constant of peptides . To determine [PI] the following equation was used: PL] was determined as described above  as a mono exponential decay (GraphPad Prism 6) using the equation belowI(t) is the signal intensity or integral at CPMG filter t (in seconds), I(0) is the signal intensity when t = 0, and T2 is the time constant of decay.Spectra were acquired for each sample of spy alone and spy in the presence of protein (wild-type and SNP mutants) at 298 K, and 80 scans using a CPMG pulse sequence with varying relaxation delays of 0.05, 0.1, 0.2, 0.4, and 0.8 s. Protein was used at 5 μM and spy was used at 100 μM in buffer containing 20 mM HEPES pH 8, 50 mM NaCl, 1 mM DTT, 20 % DFurther information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


We conducted aggregation using the well of the well system and confirmed that aggregation using two or three embryos was useful for obtaining blastocysts. Aggregated embryos significantly improved developmental competence, including blastocyst formation rate, blastomere number, ICM/TE ratio, and cellular survival rate, compared to non-aggregated embryos. Investigation into the relationship between embryo aggregation and stress conditions revealed that mitochondrial function increased, and oxidative and endoplasmic reticulum (ER)-stress decreased compared to 1X (non-aggregated embryos) blastocysts. In addition, 3X (three-embryo aggregated) blastocysts increased the expression of pluripotency, anti-apoptosis, and implantation related genes, and decreased expression of pro-apoptosis related genes. Therefore, these findings indicate that embryo aggregation regulates in vitro stress conditions to increase developmental competence and contributes to the in vitro production of high-quality embryos and the large-scale production of transgenic and chimeric pigs.Embryo aggregation is a useful method to produce blastocysts with high developmental competence to generate more offspring in various mammals, but the underlying mechanism(s) regarding the beneficial effects are largely unknown. In this study, we investigated the effects of embryo aggregation using 4-cell stage embryos in  The ER  culture . The acc culture . Studies culture . Reactiv culture . ROS-ind culture . To over culture , glutath culture , and fet culture , to enha culture . Further culture . For insl number . Other sl number .oct4 gene expression in blastocysts , unless otherwise noted.β-mercaptoethanol, 0.57 mM cysteine, 10 ng/mL epidermal growth factor, 10 IU/mL pregnant mare serum gonadotropin (PMSG) and 10 IU/mL human chorionic gonadotropin (hCG) in a 4-well multi-dish  for 22 h at 38.5 °C in 5% CO2 in air. After the first 22 h of IVM, the COCs were transferred in to fresh maturation medium without PMSG and hCG for additional 22 h at 38.5 °C in 5% CO2 in air.Porcine ovaries were collected from a nearby local abattoir in 0.9% saline containing 50 µg/mL streptomycin sulfate and 75 µg/mL potassium penicillin G at 38.5 °C within 2 h. Cumulus-oocyte complexes (COCs) were retrieved from follicles (3–7 mm in diameter) using an 18-gauge needle fixed to a disposable 10 mL syringe. COCs were washed three times with Tyrode’s Albumin Lactate Pyruvate-HEPES medium . Next, 52⋅2H2O, 0.1 mM MgSO4⋅7H2O and 0.01% polyvinyl alcohol (PVA) (2 in air. Activated oocytes were washed and cultured in PZM-3 at 38.5 °C in 5% CO2 in air.Metaphase II (MII) oocytes were placed in a 1 mm gab wire chamber  added with 280 mM mannitol containing 0.5 mM HEPES, 0.1 mM CaClol (PVA) . MII ooc2⋅2H2O, 20 mM Tris , 11 mM glucose, and 5 mM sodium pyruvate, with no antibiotics. MII oocytes were washed three times in mTBM containing 2.5 mM caffeine sodium benzoate and 1 mg/mL bovine serum albumin (BSA), and 10–15 oocytes were placed into 48 µL droplets of IVF medium under mineral oil pre-equilibrated at 38.5 °C in 5% CO2 in air. To prepare the spermatozoa using the swim-up method before fertilization, freshly ejaculated semen was washed three times with sperm washing medium . After washing, 2 mL of sperm washing medium was gently added to the spermatozoa pellet and incubated for 15 min at 38.5 °C in 5% CO2 in air. After incubation, the supernatant was washed with mTBM, and then resuspended with 1 mL of mTBM. Then, 2 µL of diluted spermatozoa was added to 48 µL of mTBM containing 10–15 oocytes to a final concentration of 1.5 × 105 spermatozoa/mL. Oocytes were co-incubated with the spermatozoa for 6 h at 38.5 °C in 5% CO2 in air. After 6 h, oocytes were stripped by gentle pipetting and transferred to PZM-3 for culture at 38.5 °C in 5% CO2 in air.IVF was carried out as described previously . IVF was2 in air . Zona-free 4 cell stage embryos were washed DPBS containing 4mg/mL BSA and placed in 1% DiI or DiO for 10 min, and then washed with PZM-3 and cultured in aggregation microwells with the darning needle. Fluorescence was observed under a fluorescence microscope .Blastocysts were fixed in 4% paraformaldehyde overnight at 4 °C and washed three times in DPBS with 0.1% PVA (DPBS-PVA). The fixed blastocysts were treated with DPBS containing 0.5% Triton X-100 for 1 h at room temperature (RT), then washed in DPBS-PVA. Next, the blastocysts were incubated in DPBS-PVA supplemented with 1 mg/mL BSA (DPBS-PVA-BSA) at 4 °C overnight and were stored with 10% normal goat serum for 1 h at RT. Primary antibody was used mouse monoclonal anti-Cdx2  for overnight at 4 °C. After washed three times with DPBS-PVA-BSA for 10 min and incubated for 1 h at RT with conjugated secondary antibodies, Alexa-Fluor-488-labeled goat anti-mouse IgG (1:200). After washed three times in DPBS-PVA-BSA for 10 min and stained with 2 µg/mL 4′, 6′-diamidino-2-phenylindole (DAPI). DAPI-labeled or Cdx2-positive nuclei were observed using a fluorescence microscope (Olympus).in-situ cell death detection kit . The blastocysts were washed three times in DPBS-PVA and fixed in 4% paraformaldehyde overnight at 4 °C. Fixed blastocysts were permeabilized in DPBS containing 0.5% Triton X-100 at RT for 1 h. Subsequently, blastocysts were washed three times with DPBS-PVA and stained with fluorescein-conjugated dUTP and terminal deoxynucleotidyl transferase for 1 h at 38.5 °C. The blastocysts were washed three times with DPBS-PVA and mounted on clean glass slides with DAPI. DAPI-labeled or TUNEL-positive nuclei were observed under a fluorescence microscope (Olympus).TUNEL assay was carried out using an The MitoTracker and JC-1 staining were modified carried out as described previously . BlastocMeasurement of ROS levels in embryos were carried out as described previously . Intrace2+), oligo(dT)20, 10 mM mixture of dNTPs and 10 U of the RNase inhibitor ReverTra Ace . The following PCR conditions were used: 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s, followed by extension at 72 °C for 5 min. qRT-PCR were performed with SYBR premix Ex Taq (Takara Bio Inc.) using Mx3000P QPCR system . The sample delta Ct (SΔCT) value was calculated from the difference between the Ct values of GAPDH the target genes. The relative gene expression levels between the samples and the controls were determined using the formula 2SΔCT−CΔCT)−(. The primers used in the present study are listed in Poly(A) mRNAs were extracted using the Dynabeads mRNA Direct kit (Invitrogen) from 20 blastocysts according to the manufacturer’s protocol. The resulting poly(A) mRNAs were reverse transcribed in 20 µL reactions containing 5 × RT buffer (containing 25 mM MgAF304202) to amplify a 151-base pair. Melting curve was analyzed to verify the specificity of the PCR products, followed by electrophoresis to determine the product size. As an external standard, the PCR product of the corresponding gene was cloned into a vector using the Zero Blunt TOPO PCR cloning kit (Invitrogen). The product was sequenced for confirmation before use.Analysis of mitochondrial DNA copy number was carried out as described previously . The mitP-values less than 0.05 were considered to indicate statistical significance.The blastocyst formation rate (blastocyst formation number per cultured embryos) and proportion of blastocysts by diameter  were recorded as the percentage. The cell numbers within the blastocysts were counted by DAPI- labeled or Cdx2- positive nuclei . Apoptosis  was recorded as the percentage. The number of independent replicates (Re) of each experiment is shown in the figure legends. Data are expressed as the mean ± standard error of the mean (SEM). Data were compared using analysis of variance (ANOVA), followed by Duncan’s multiple range test using SigmaStat Software . To confirm that the PA embryos could aggregate, we cultured zona-intact , two-embryo aggregated 2X) and three-embryo aggregated (3X) using only 4-cell stage embryos, respectively. The zona pellucida of 4-cell stage embryos was removed by treatment with acidic Tyrode’s solution, and then differentially incubated with DiI (red) or DiO (green) for membrane staining. Embryo aggregation was observed in the morula and blastocyst stages derived from 2X and 3X embryos –2L. Fluo and threTo investigate the effect of aggregation on the developmental competence of porcine embryos, PA embryos  were tested for aggregation in microwells. Compared to the NC, 1X embryos showed no detrimental effect on blastocyst formation upon removal of the zona pellucida. Meanwhile, 2X and 3X aggregated embryos showed significant increases in blastocyst formation rate  and blas2DCFDA staining and fluorescence microscopy. ROS levels were markedly lower in the 3X than 1X group  but restored by treatment with tauroursodeoxycholic acid  . In thisstocysts . Previou unknown . Similar2+ signaling pathway, energy metabolism, and cellular survival  involved the ER stress-induced apoptosis signaling pathway, including the Bcl-2 family via the release of cytochrome C . (D) Proportion by blastocysts diameter in the indicated groups.  Immunocytochemistry of Cdx2/DAPI using blastocysts developed in the indicated groups. Merged images between DAPI (blue) and Cdx2 (green) signals are shown. Bar = 50 um. (G-J) Quantification of the total, ICM, TE cell numbers, and ICM/TE ratios in the indicated groups. (*P ¡ 0.05).  TUNEL assay using blastocysts developed in the indicated groups. Merged images (light green) between DAPI (blue) and TUNEL  signals are shown. Bar = 50 um. (M) Quantification of proportion of apoptotic cells in the indicated groups. (*P ¡ 0.05). For all panels, n indicates number of embryos examined. Re = 3. 1X; one zona-free embryo, 3X; three zona-free embryos Representative photographs of blastocysts developed from the indicated group for aggregation. Bar = 100 um. (C) Blastocyst formation rates in the indicated groups. .Data are the mean ± SEM, and values with different superscript letter within a column differ significantly .Data are the mean ± SEM, and values with different superscript letter within a column differ significantly .Data are the mean ± SEM, and values with different superscript letter within a column differ significantly .Data are the mean ± SEM, and values with different superscript letter within a column differ significantly .Data are the mean ± SEM, and values with different superscript letter within a column differ significantly .Data are the mean ± SEM, and values with different superscript letter within a column differ significantly .Data are the mean ± SEM, and values with different superscript letter within a column differ significantly .Data are the mean ± SEM, and values with different superscript letter within a column differ significantly (Click here for additional data file.10.7717/peerj.8143/supp-11Data S1Raw data applied for data analyses and preparation for Figure 3B, 3C, 3E and 3G; Figure 4B, 4D, 4E and 4G; Figure 5A, 5B and 5C; Figure 6A, 6B and 6C; Supplementary figure 1B, 1C, 1E and 1G.Click here for additional data file.


Obesity and nonalcoholic steatohepatitis (NASH) are well-known risk factors of hepatocellular carcinoma (HCC). The lipid-rich environment enhances the proliferation and metastasis abilities of tumor cells. Previous studies showed the effect of the ubiquitin–proteasome system (UPS) on tumor cell proliferation. However, the underlying mechanism of UPS in regulating the proliferation of lipid-rich tumor cells is not totally clear.PSMD1 and PSMD2), which regulate HepG2 cells proliferation via modulating cellular lipid metabolism. Briefly, the knockdown of PSMD1 and/or PSMD2 decreases the formation of cellular lipid droplets, the provider of the energy and membrane components for tumor cell proliferation. Mechanically, PSMD1 and PSMD2 regulate the expression of genes related to de novo lipid synthesis via p38-JNK and AKT signaling. Moreover, the high expression of PSMD1 and PSMD2 is significantly correlated with poor prognosis of HCC.Here, we identify two proteasome 26S subunits, non-ATPase 1 and 2 (PSMD1 and PSMD2 promote the proliferation of HepG2 cells via facilitating cellular lipid droplet accumulation. This study provides a potential therapeutic strategy for the treatment of lipid-rich tumors.We demonstrate that  Hepatocellular carcinoma (HCC) is one of the most frequent causes of cancer-related deaths worldwide . The chrPSMD1 and PSMD2 are two subunits of the 19S regulatory complex of the 26S proteasome [PSMD1 and/or PSMD2 is able to suppress tumor cell proliferation [PSMD1 and PSMD2 are LD-related proteins in several species such as humans, mice, and C. elegans [PSMD1 and PSMD2 in cellular lipid metabolism are unclear.The ubiquitin–proteasome system (UPS) is a huge and complex system that regulates the degradation of up to 80% of cellular proteins, consisting of ubiquitin (Ub), Ub-activating enzymes , Ub-conjugating enzymes , Ub-protein ligases , deubiquitinating enzymes , and the heart of the system, the 26S proteasome complex –25. Prevoteasome –38. Receferation –41. Many elegans –48. HowePSMD1 and PSMD2 in cell proliferation and cellular lipid metabolism. HepG2 cells were derived from 15-year-old white liver cancer tissue and were utilized in the study about hepatocyte metabolism. Firstly, we showed that the expression levels of PSMD1 and PSMD2 affected cell proliferation and apoptosis. The knockdown of PSMD1 and PSMD2 inhibited cell proliferation and promoted cell apoptosis, and overexpression of PSMD1 and PSMD2 showed the opposite effects. Furthermore, the expression of PSMD1 and PSMD2 affected several proliferation and apoptosis related molecules. Because cellular lipid content is associated with cell proliferation and apoptosis, we further investigated the effects of PSMD1 and PSMD2 expression on cellular lipid metabolism. The knockdown of PSMD1 and PSMD2 decreased the content of cellular lipids. On the contrary, the overexpression of PSMD1 and PSMD2 promoted lipid formation and increased the cellular lipid content. Mechanically, PSMD1 and PSMD2 inhibition suppressed fatty acid and lipid synthesis by downregulating SREBF1 and PPARγ via ASK-p38-JNK and AKT signaling. Moreover, through bioinformatic analysis, high expression levels of PSMD1 and PSMD2 were significantly correlated with poor prognosis of liver hepatocellular carcinoma. Therefore, the high expression levels of PSMD1 and PSMD2 probably enhanced hepatocellular carcinoma tumor cell proliferation. PSMD1 and PSMD2 could be potential therapeutic targets for this type of tumor.In the present study, we chose a hepatocellular carcinoma cell line, HepG2, to investigate the roles of 2. Cells were transfected with Lipo8000™ Transfection Reagent . HepG2 cells were seeded on the cell slide in a 6-well plate and transfected with the plasmid vector in accordance with the transfection reagent instructions.HepG2 and Huh7 cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences . Cells were cultured with Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  at 37 °C in a humidified atmosphere of 5% COFor oleic acid treatment, a 20 mM oleic acid (LPS free)-phosphate buffer saline (PBS) mixture and 20% FA-free bovine serum albumin (BSA) medium were prepared, and both media were heated in a 70 °C water bath for 30 min. Finally, the media were mixed. The 10 mM oleic acid–BSA mixture was added to the cell cultural medium at 1:49 (v:v). To identify the best treatment time, a time course was performed. The cells were treated with 200 μM oleic acid (OA) for 0, 1, 2, 3, 4, 5, and 6 h respectively, and then were stained by BODIPY to indicate the cellular LDs. The images showed that cellular LDs were able to be observed at 2 h after OA treatment. Many LDs formed “grape-like” structures at 5 h after OA treatment, and additionally, the image from the 6 h treatment showed little difference from the image from the 5 h treatment  was added to each well and cells were cultured for another 4 h. Furthermore, 100 μL Formazan solution was added into each well and the optical density (OD) at 570 nm was measured on a microplate reader .HepG2 cell proliferation was detected using MTT Cell Proliferation and Cytotoxicity Assay Kit . Briefly, cells were seeded at a density of 4 × 103/well in 96-well plates. The cells were treated with 200 μM oleic acid for 0 h, 6 h, 12 h and 24 h. Then 10 μL CCK8 was added to each well and cells were cultured for another 4 h. The optical density (OD) at 450 nm was measured on a microplate reader .HepG2 and Huh7 cells activity after oleic acid treatment was detected by Cell Counting Kit-8 (CCK8) kit . Briefly, cells were seeded at a density of 4 × 10Cells were collected and fixed in 70% ice-cold ethanol at 4 °C overnight. Then, the cells were centrifuged and washed with PBS. After incubation with 100 μL RNase A (0.1 mg/mL) for 30 min at 37 °C, 2 μL propidium iodide  was add to the cells and incubated for another 30 min at room temperature in the dark. Finally, cell cycle profiling was performed using a FACSCalibur Flow Cytometer , and the data were analyzed using ModFit software (Verity Software House).At 48 h after transfection, the percentage of apoptotic cells was measured using an Annexin V fluorescein isothiocyanate (FITC) apoptosis detection kit  and analyzed by flow cytometry  in accordance with the manufacturer’s protocol.Cells seeded in 12-well plates were cultured to 50% confluence and then transfected. Forty-eight hours after transfection with siRNAs or overexpression plasmid, the cells were fixed and stained with a BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 594  in accordance with the instructions. A fluorescence microscope  was used to capture three randomly selected fields to visualize the number of EdU-positive cells.PSMD1 and PSMD2 were obtained from Ribobio . The sequence of the siRNA against the PSMD1 coding region was 5′-GAGAAAGACAGUGACUCGAUGGAAA-3′, and that of the siRNA against the PSMD2 coding region was 5′- CCCAAGGUGCCUGAUGACAUCUACA-3′ [siRNA oligos against CUACA-3′ . The expPSMD1 and PSMD2 were obtained from Ribobio . Cells were seeded onto 6-well plates in DMEM supplemented with 10% FBS at 37 °C in a humidified atmosphere of 5% CO2. Cells at 70–80% confluence were transfected with 10 µL of siRNA using Lipo8000™ Transfection Reagent . For the overexpression assay, 4 µg of the constructed plasmid was transfected with 10 µL of Lipo8000™ Transfection Reagent . All transfections were performed in triplicate, and the data are presented as the mean ± standard deviation (SD).The siRNAs targeting ® 480 Real-Time System (Roche) and QuantStudio 6 Flex Real-Time PCR System . The primer sequences used for qRT-PCR are listed in Additional file GAPDH and ACTB, the most frequently used housekeeping genes, were chosen as candidates. Then, the stability of the candidate reference genes was analyzed through standard curve and geNorm methods . RNA concentrations were determined using spectrophotometry with a NanoDrop 2000 (Thermo Fisher Scientific). A total of 1 μg of RNA was then subjected to reverse transcription to cDNA using a Reverse Transcription Kit . qRT-PCR was performed with SYBR Green I Real-Time PCR Master Mix  on a LightCycler’s study . The relPSMD1 , anti-PSMD2 , anti-ASK1 , anti- p-ASK1 , anti-AKT , anti-p-AKT , anti-p38 , anti-p-p38 , anti-JNK1/2 , anti-p-JNK1/2 , anti-GAPDH , anti-Tubulin , HRP-labeled Goat Anti-Rabbit IgG (H + L) , and HRP-labeled Goat Anti-Mouse IgG (H + L) .Western blotting was performed as reported previously . The folCells were seeded on slides on a 24-well plate and were cultured for 24 h. The slides were fixed in 4% paraformaldehyde  for 15 min at room temperature. The slides were stained with BODIPY 493/503  for 45 min at 37 °C and were then stained with DAPI  for 10 min at 37 °C. After washing three times with PBS for 10 min each, the slides were sealed with an anti-fluorescent quenching solution  for use in a confocal laser scanning microscope  observation.PSMD1, PSMD2, and PLIN2 expression vectors  were co-transfected into HepG2 cells. After 48 h, the cells were treated with 200 μM OA for another 6 h. Then, the cells were fixed and then sealed with an anti-fluorescent quenching solution for confocal laser scanning microscope observation. The fluorescence plot analysis was performed by ImageJ software according to the instructions (https://imagej.en.softonic.com/). For the co-localization of PSMD1/PSMD2 and newly formed LDs, Livedrop was introduced to this experiment. The Livedrop [The fluorescence labelling Livedrop  has beenhttp://gepia.cancer-pku.cn/). The prognosis analysis and gene expression analysis were performed according to the instructions of the creator of this database [Survival predication was performed using the GEPIA database  in the siPSMD1, siPSMD2, and siPSMD1 + siPSMD2 group cells, while CASP3, CASP8, CASP9, and FAS were upregulated significantly (p < 0.05). To validate this result, the EdU assay was performed to detected cell proliferation. The number of proliferating cells was approximately 60% of the total cells in the control group, whereas the percentages were about 40%, 39%, and 38% in the siPSMD1, siPSMD2, and siPSMD1 + siPSMD2 groups, respectively . Usually, cells form the fatty acid and lipid droplets de novo. The cellular LDs were marked by BODIPY493/503, a specific neutral lipid targeting dye, and the numbers and sizes of LDs in different cell groups were analyzed based on the images captured by the confocal laser scanning microscope  and neutral lipid synthesis-related genes . We first investigated the effect of the suppression of PSMD1 and PSMD2 expression on these genes. The efficiency of interference was reflected by the mRNA levels of PSMD1 and PSMD2 detected by qPCR , which was in contrast to the results of the interference experiment. This result suggests that PSMD1 and PSMD2 may regulate SEIPIN expression.The changes in LD number and size prompted us to investigate the effects of PCR Fig. a, and ths p < 0.0, Fig. 5bPSMD1 and PSMD2 regulate FA and lipid synthesis-related genes, we detected two classical pathways, the ASK1-p38-JNK and AKT signaling pathways. We detected the phosphorylated ASK1, p38, and JNK1/2 levels and found that the p-ASK1, p-p38, and p-JNK1/2 levels were decreased in interference group cells compared with control cells . The LDs can carry proteins from the ER and recruit proteins through the ARF-COPI system. Therefore, we further investigated the source of LD localized LDs Fig. g, h. ThiPSMD1 or PSMD2 on the prognosis of liver hepatocellular carcinoma (LIHC), we analyzed the expression levels of PSMD1 and PSMD2 in LIHC via the GEPIA  database (http://gepia.cancer-pku.cn/). The data showed that PSMD1 and PSMD2 are highly expressed in LIHC  [SIRT7 controls hepatic lipid formation and accumulation via regulating the ubiquitin–proteasome pathway, and SIRT7 knockout mice have shown resistance to high-fat-diet induced fatty liver [PSMD1 and PSMD2, which could affect the cellular lipid content. We detected the FA and lipid synthesis enzyme expression levels during PSMD1/PSMD2 knockdown or cell overexpression, and the results indicated that PSMD1 and PSMD2 indeed affect the expression levels of FASN, SCD1, SREBF1, DGAT, and PPARγ  and newly formed LDs (labelled by Livedrop-EGFP) were found to be related to the localization of PSMD1 and PSMD2  , and adds PLIN2) . Moreoves PLIN2) , and adds PLIN2) . Furtherty liver . Moreovety liver . All of ARγ Fig. . Althoug process –85. Addi process –93. We fl growth , 57. TheARγ Fig. , which sPSMD1 and PSMD2 could regulate the cellular lipid content, thereby affecting cell proliferation progression. Mechanistically, PSMD1 and PSMD2 regulate FA and lipid synthesis-related gene expression through ASK1-p38-JNK and AKT signaling. The molecular mechanism of PSMD1/PSMD2 regulating cell proliferation is illustrated in Fig. PSMD1/PSMD2 regulatory mechanism in HCC cell proliferation and provides a potential novel therapeutic strategy for lipid-rich tumor.In the present study, we identified that Additional file 1: Fig. S1. Time course experiment of oleic acid medium treatment. A The cells were treatment with 200 μM oleic acid. The cellular lipid droplets were imaged at 1 h, 2 h, 3 h, 4 h, 5 h and 6 h after oleic acid treatment. B, C The cell activity of Huh7 cells and HepG2 cells was detected by CCK8 method after 200 mM oleic acid medium treatment for 6 h, 12 h and 24 h.Additional file 2. Additional tables.Additional file 3: Fig. S2.PSMD1 and PSMD2 expression level regulates the number and size of cellular lipid droplets in Huh7 cells. The Huh7 cells were seeded on the slide in a 24-well plate. Then, the cells were transfected with PSMD1/PSMD2 or NC siRNAs for 48 h for knockdown (A–C), or transfected with PSMD1/PSMD2 expression vector or NC vector for 48 h for overexpression (D–F). Subsequently, the cells were treated with 200 μM oleic acid for another 6 h. Then, the cells were fixed and stained by BODIPY493/503 and DAPI for observation by microscope.  The number of cellular LDs from different groups of cells. ImageJ software was used for the analysis. The statistical significance of differences between means was assessed using an unpaired Student’s t-test  vs. NC.  The size of cellular LDs of different groups of cells. ImageJ software was used for the analysis. The statistical significance of differences between means was assessed using an unpaired Student’s t-test  vs. NC.


Phaseolus vulgaris cv. “gold bean” with an isolation protocol including ion exchange chromatography on DEAE-cellulose (Diethylaminoethyl-cellulose), affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-sepharose (Sulfopropyl-sepharose), and gel filtration by FPLC (Fast protein liquid chromatography) on Superdex 75. It dose-dependently inhibited trypsin with an IC50 value of 0.4 μM, and this activity was reduced in the presence of dithiothreitol in a dose- and time-dependent manner, signifying the importance of the disulfide linkage to the activity. It inhibited [methyl-3H] thymidine incorporation by leukemia L1210 cells and lymphoma MBL2 cells with an IC50 value of 2.3 μM and 2.5 μM, respectively. The inhibitor had no effect on fungal growth and the activities of various viral enzymes when tested up to 100 μM.A 17.5-kDa trypsin inhibitor was purified from  Momordica cochinchinensis  thymidine incorporation by leukemia L1210 cells and lymphoma MBL2 cells.Phaseolus vulgaris cv. “gold bean” from Mainland China (260 g) was made by homogenizing them in distilled water (6 mL/g). The homogenate was then centrifuged . The supernatant was collected and loaded on a 5 × 20 cm column of DEAE-cellulose  in 10 mM Tris-HCl buffer (pH 7.4). Following removal of unadsorbed proteins (fraction D1), the column was eluted sequentially with 0.2 M NaCl and 1 M NaCl in the Tris-HCl buffer. Fraction D2 eluted with 0.2 M NaCl was dialyzed and then chromatographed on a 5 × 15 cm column of Affi-gel blue gel  in 10 mM Tris-HCl buffer (pH 7.4). The unadsorbed proteins (fraction B1) were dialyzed against 10 mM NH4Ac buffer (pH 5) and applied on a 2.5 × 20 cm column of SP-sepharose . After elution of unadsorbed proteins (fraction S1), the column was eluted with a 0–1 M NaCl concentration gradient in the NH4Ac buffer. The first adsorbed fraction (S2) was then subjected to gel filtration on a Superdex 75 HR 10/30 column  in 0.2 M NH4HCO3 buffer (pH 8.5). The second absorbance peak (SU2) represented purified trypsin inhibitor (GBTI).A water extract of gold beans The assay for trypsin inhibitory activity was carried out by addition of test sample (20 μL) to 160 μL of a 1% casein solution in 0.1 M Tris-HCl buffer (pH 7.4). Trypsin (20 μL of a 0.5 mg/ml solution) was then added and the mixture was incubated at 37 °C for 15 min before 0.4 mL 5% trichloroacetic acid was added to terminate the reaction. After centrifugation, the absorbance of the supernatant, which reflects the amount of casein fragments, was measured at 280 nm .The purified trypsin inhibitor was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis  for molecular mass determination in accordance with the procedure of Laemmli and Favre . Gel filThe purified trypsin inhibitor was incubated in a water bath at different temperatures from 20 °C to 100 °C for 15 min, then immediately chilled on ice. The reaction mixture was then subjected to the assay of trypsin inhibitory activity.The purified trypsin inhibitor (2.5 μM) was incubated with dithiothreitol (DTT) at the final concentration of 2.5, 10, and 40 mM for 5, 20, and 80 min at 37 °C, respectively. For comparison, soybean trypsin inhibitor purchased from Sigma Chemical Co.,  (2.5 μM) was similarly treated. The reaction was terminated by adding iodoacetamide at twice the amount of thiol functions at each DTT concentration. The remaining trypsin inhibitor activity was measured at pH 7.4, as described above. The highest iodoacetamide concentration used in the test was devoid of any effect on the activity of trypsin and the trypsin inhibitory activity of purified trypsin inhibitor and soybean trypsin inhibitor .2. Cells (1 × 104) in their exponential growth phase were seeded into each well of a 96-well culture plate  and incubated for 3 h before addition of the trypsin inhibitor. Incubation was carried out for another 48 h. Radioactive precursor, 1 μCi , was then added to each well and incubated for 6 hrs. The cultures were then harvested by a cell harvester. The incorporated radioactivity was determined by liquid scintillation counting  for 40 min at 37 °C. After 40 min, the reaction was stopped by heating at 100 °C for 2 min. Then the reaction mixture was analysed by SDS-PAGE. If SARS CoV protease is inhibited by the test sample, there is only one band, which is the intact substrate, shown in SDS-PAGE .Mycosphaerella arachidicola and Fusarium oxysporum was carried out in 100 mm × 15 mm Petri dishes containing 10 ml of potato dextrose agar. After the mycelial colony had developed, sterile blank paper disks (0.625 cm in diameter) were placed at a distance of 0.5 cm away from the rim of the mycelial colony. An aliquot (15 μL) of the purified trypsin inhibitor was added to a disk. The plates were incubated at 23 °C for 72 h until mycelial growth had enveloped the disks containing the control and had formed crescents of inhibition around the disks containing samples with antifungal activity  thymidine incorporation by leukemia L1210 cells and lymphoma MBL2 cells was inhibited with an IC50 value of about 2 μM. Mycelial growth in Fusarium oxysporum and Mycosphaerella arachidicola were unaffected. The activities of HIV-1 reverse transcriptase, HIV-1 integrase and SARS coronavirus proteinase were unaltered after exposure to the trypsin inhibitor.A 17.5-kDa trypsin inhibitor was purified from