To better understand how NO3− and NH4+ differentially affect root system architecture, we analyzed the expression profiles of microRNAs and their targets in poplar roots treated with three forms of nitrogen S1 (NO3−), S2 , and S3 (NH4+) via RNA sequencing.Nitrate (NONFYA3, a target of upregulated ptc-miR169i and ptc-miR169b, was downregulated in S1 vs. S2, while NFYA1, a target of upregulated ptc-miR169b, was downregulated in S3 vs. S2 and probably played an important role in the changes in root morphology observed when the poplar plants were treated with different N forms. Furthermore, the miRNA-target pairs ptc-miR169i/b-D6PKL2, ptc-miR393a-5p-AFB2, ptc-miR6445a-NAC14, ptc-miR172d-AP2, csi-miR396a-5p_R + 1_1ss21GA-EBP1, ath-miR396b-5p_R + 1-TPR4, and ptc-miR166a/b/c-ATHB-8 probably contributed to the changes in root morphology observed when poplar plants were treated with different N forms.The results revealed a total of 709 miRNAs. Among them, 57 significantly differentially expressed miRNAs and 28 differentially expressed miRNA-target pairs showed correlated expression profiles in S1 vs. S2. Thirty-six significantly differentially expressed miRNAs and 12 differentially expressed miRNA-target pairs showed correlated expression profiles in S3 vs. S2. In particular, These results demonstrate that differentially expressed miRNAs and their targets play an important role in the regulation of the poplar root system architecture by different N forms.The online version contains supplementary material available at 10.1186/s12870-022-03482-3. In mmonium NH+ are therphology –7. In PoP. nigra  and Arabthaliana , NH4+ cathaliana . Despitethaliana , 8, 9, lArabidopsis roots, NO3− (5 mM) inhibits miR167 expression while promoting the expression of its target ARF8 (AUXIN RESPONSE FACTOR 8), which promotes lateral root initiation and emergence [Oryza sativa), miR166 targets RDD1(rice Dof daily fluctuations 1), which is related to NH4+ uptake and transport, leading to a change in root morphology [3− and NH4+ and in turn regulate their target genes and affect the plant root system architecture [miRNAs are highly conserved, endogenous, noncoding small RNAs with lengths of approximately 18–25 nucleotides. miRNAs inhibit posttranscriptional gene expression by inducing the cleavage of target genes or weakening translation, affecting organismal morphogenesis, development, and adaptability to environmental changes –14. In rmergence , 15. In itecture , 15, 19.Populus × canescens and P. simonii × P. nigra) as well as the regulation of distinct genes in the presence of different N forms [3− absorption rates in different sections of poplar root tips [3− and NH4+ or both).Poplar, which presents a large demand for N fertilizer, has become a model system for research on the molecular mechanisms of woody plant root growth, development, and responses to the environment . Some pr N forms . Our preoot tips . HoweverP. × canescens when treated with 1 mM NO3− (S1), 500 μM NH4NO3 (S2) or 1 mM NH4+ (S3) for 21 days. The primary goal of this work was to distinguish the morphological changes in poplar roots and analyze the potential miRNA-target pair regulation mechanisms of root morphological characteristics of poplar trees under different N forms. To achieve this goal, root length, lateral root initiation and lateral root density were measured. The results showed spatial variability in poplar root length and dry weight in the presence of different N forms. Through miRNA sequencing, degradome sequencing and transcriptomic sequencing, several candidate miRNA-target pairs associated with morphological changes in poplar roots were identified. The results of this work provide new ideas for the regulatory mechanisms whereby miRNA-mRNA regulatory networks change the root system architecture in response to different N forms, facilitating our understanding of the mechanism of root system architecture regulation in the presence of different N forms mediated by miRNA-target pairs.In this study, using a hydroponic culture system, we monitored the root system architecture and molecular changes in P. × canescens roots showed significantly different phenotypes after 21 days of long-term hydroponic cultivation with 1 mM NO3− (S1), 500 μM NH4NO3  or 1 mM NH4+ (S3). When the roots were grown in medium containing S1, root growth was significantly promoted compared to that in the presence of S2, whereas when the roots were grown in medium containing S3, root growth was significantly inhibited compared to that in the presence of S2. The length of the roots in S3 was 64% of that in S1  could be fitted by linear equations in both qRT–PCR and miRNA-seq. The slopes were 0.34 in S1 vs. S2  in qRT–PCR was positively correlated with miRNA-seq in S1 vs. S2 and S3 vs. S2, indicating that miRNA-seq results were reliable.To validate the miRNA sequence results, we selected twenty-two miRNAs in S1 vs. S2 and fifteen miRNAs in S3 vs. S2 for qRT–PCR  sequences that were mapped to 6 812 869 unique transcript reads were successfully mapped against the AAP7 (an amino acid transmembrane transporter), which is targeted by gra-MIR8654c-p3_2ss12TG18AG family members, is responsible for the transport of amino acids. Several targets, including NFYA1/3 (nuclear transcription factor Y subunit A), ARFs (auxin response factors), AP2/EREBP (APETALA2/ethylene-responsive element binding protein) family members and NAC (NAC transcription factor) transcription factors, which belong to the miR169, miR160, miR172 and miR6445 families, accounted for the largest proportion of targets involved in the RNA-mediated regulation of transcription  and stu-miR166b_R-1_1ss19CT and its target eIF (eukaryotic translation initiation factor) (Potri.007G119200.2) were selected for transient coexpression experiments. The NFYA3 transcript level was significantly decreased after NFYA3 and ptc-miR169i_1ss15TA coexpression in N. benthamiana leaves for 2 days, and the expression of the target gene eIF was significantly decreased under eIF and stu-miR166b_R-1_1ss19CT coexpression; thus, the expression levels of the target genes NFYA3 and eIF were decreased considerably by ptc-miR169i_1ss15TA and stu-miR166b_R-1_1ss19CT, respectively, in comparison with the expression levels observed under transient expression of the NFYA3 and eIF target genes alone , ptc-miR393a-5p and its target AFB2 (AUXIN SIGNALING F-BOX2), ptc-miR6445a and its target NAC14, ptc-miR172d and its target AP2, ath-miR396b-5p_R + 1 and its target TPR4 (topless-related protein 4-like), and csi-miR396a-5p_R + 1_1ss21GA and its target EBP1 (ERBB-3 BINDING PROTEIN 1). Four miRNA-target pairs in S3 vs. S2 had close relationships with the regulation of the poplar root system architecture . In particular, NFYA3, a target of upregulated ptc-miR169i and ptc-miR169b, was downregulated in S1 vs. S2, while NFYA1, a target of upregulated ptc-miR169b, was downregulated in S3 vs. S2, indicating that miR169-NFYA pairs probably had an important and complex regulatory effect on poplar root system architecture under different N forms.A BLASTN search of 3− or NH4+ is used as the sole N source [3− to plants mainly promotes the elongation of lateral roots, whereas the supply of NH4+ increases the initiation and branching of lateral roots [3−- or NH4NO3-treated P. simonii × P. nigra seedlings was greater than that of NH4+-treated seedlings after 21 days of treatment [Arabidopsis, the local supply of NH4+ increased lateral root initiation and lateral root branching [3− - or NH4NO3-treated roots were greater than those of NH4+-treated plants, and NH4+ treatment resulted in earlier lateral root initiation than that observed under NO3− treatment. These results suggest that poplar roots display high plasticity in response to different N forms. We propose that the greater root length observed under the influence of NO3− could be a result of the increased supply of NO3− due to the upregulation of nitrate transporters (NRTs) in NO3−-treated roots (S3), since NRTs are NO3− transporters that enhance nitrate-dependent root elongation [4+ treatment can have adverse effects on plants, including altered root metabolism, plant ion imbalances, and foliar oxidative stress [4+ supplied as the sole nitrogen source would cause toxic symptoms and inhibit root growth in many plant species [4+ treatment.Plant roots display high plasticity in response to different N forms, and the morphological characteristics of plant roots are distinctly different when NON source , 6, 24. al roots , 25, 26.ranching , 28, in ongation . On the e stress , leading3− and NH4+ libraries, respectively. These results suggested that NO3− treatment elicits stronger miRNA responses than NH4+ treatment. The difference may reflect the fact that NO3− supplied during long-term cultivation (21 days) can trigger a markedly different miRNA response in P. × canescens, which is consistent with previous research in rice [3−-treated plants (16 miRNAs) was greater than that in NH4+-treated plants (11 miRNAs) [3− treatment was greater than that under NH4+ treatment (data not shown), which was consistent with previous findings in P. simonii × P. nigra [3− treatment were greater than the 126 differentially expressed genes identified under NH4+ treatment in P. simonii × P. nigra [3− is an important signaling molecule [3− treatment on miRNA expression may be more pronounced than that of NH4+ treatment.The morphological changes caused by different N forms are closely related to changes at the molecular level. To better understand the effects of different N forms on root morphology and growth, we detected the differentially expressed miRNAs and their targets in poplar roots treated with different N forms. In this study, 57 and 36 miRNAs showed differential expression patterns in the NOmolecule –32, it iNFYA transcripts play a role in the control of root growth in A. thaliana [TaNFYA-B1 promoted the expression of TANRT1.1 and TANRT2.1, increased root NO3− influx and promoted lateral root growth [NFYA3, which is homologous to TaNFYA-B1, showed a significantly downregulated expression profile in S1 vs. S2 and inhibited lateral root growth. In rice, miR169o-overexpressing plants are taller than WT plants under either normal or limiting NO3− conditions, and the predicted OsNFYA1/4 target genes display an exactly opposite expression pattern to that of osa-miR169o in response to NO3− deficiency [NFYA1 was homologous to OsNFYA4, and it showed a significantly downregulated expression profile in S3 vs. S2 and inhibited primary root elongation. These results indicate that ptc-miR169 family members and their target NFYA genes might participate in the alteration of poplar root morphology in response to different N forms. Interestingly, the expression of ptc-miR169i_1ss15TA and ptc-miR169b_R-1 exhibited another target, D6PKL2, in S1 vs. S2. D6PKL2 kinases may directly phosphorylate PIN-FORMED (PIN) proteins [d6pk mutants show defects in lateral root initiation, and this phenotype is correlated with a reduction in auxin transport in Arabidopsis [P. × canescens. Thus, a complex mechanism may modulate the expression profiles of miRNA family members and their targets.Some miRNA-target pairs were also involved in the changes in plant root morphology observed in the presence of different N forms , 7. Sevethaliana , 33, whethaliana , maize [thaliana  and poplthaliana . In wheat growth . NFYA3, ficiency . NFYA1 wproteins , and d6pbidopsis . These rTIR1 (transport inhibitor response protein 1), AFB1, AFB2, and AFB3. AtAFB3 is induced by NO3− in Arabidopsis root organs, which inhibits primary root elongation and promotes lateral root initiation [AFB2, which is homologous to an AtAFB3 sequence, was reduced in root organs after 21 days of exposure to NO3− treatment, promoting primary root elongation and inhibiting lateral root initiation. This result differed from previous findings in Arabidopsis [Arabidopsis experiment, the treatment environment and conditions were different in this experiment. Second, the AFB gene family has different functions in annual herbs and perennial woody plants. Third, in Arabidopsis, AFB3 was induced after 1 h of NO3− treatment, but miR393 was also induced after NO3− treatment, which led to the degradation of its target gene AFB3 and reset AFB3 expression to basal levels. Therefore, AFB3 was induced by NO3− treatment and degraded by miR393 to achieve negative feedback regulation [AFB2 pair has a negative feedback regulation mechanism under NO3− treatment needs to be further studied.miR393 targets transcripts that encode the auxin receptors itiation . In our bidopsis , possiblgulation . HoweverNAC14 transcription factor. A poplar homologous gene of AtNAC4 acts downstream of AFB3 to control the root response to NO3−, while nac4 mutants exhibit changes in lateral root growth in A. thaliana [TaNAC2-5A can be directly bound to the promoter regions encoding nitrate transporters and glutamine synthetase genes, which affects root growth and the nitrate influx rate [AP2/ERF transcription factor, a poplar homologous gene of AtAP2/EREBP involved in lateral root formation in Arabidopsis [EBP1. A poplar homologous gene of AtEBP1 is involved in dividing tissues during root development in Arabidopsis [TPR4. A poplar homologous gene of AtTPR4 was recruited by WUSCHEL RELATED HOMEOBOX 5 (WOX5) to inhibit target genes that promote differentiation to ensure the maintenance of stem cells [EBP1 and ath-miR396b-5p_R + 1-TPR4 miRNA-target pairs might participate in changes in P. × canescens root morphology by regulating the cell differentiation involved in NO3− treatment. The target of ptc-miR166a/b/c was ATHB8, and both miR166 and the target gene ATHB8 were upregulated in response to NH4+ treatment. A poplar homologous gene of AtATHB8 affects vascular development [The target gene of ptc-miR6445a is the thaliana , 40. In lux rate . ptc-miRbidopsis . The tarbidopsis . Anotherelopment  and may elopment , 47.P. × canescens and probably play important roles in poplar root morphogenesis. According to the present results, we propose a simple model of the response to different N forms in P. × canescens roots  were obtained by the micropropagation method. This plant stock was obtained and granted by Prof. Andrea Polle’s laboratory at the University of Göttingen in Germany. Experimental research on these plants complied institutional and national guidelines. Seedlings were grown in a climate chamber for 30 days. Thereafter, the plants were placed in plastic pots (10 L) filled with fine sand and cultivated in a growing chamber with conditions similar to those of the climate chamber. Every other day, 50 mL of modified Long Ashton (LA) solution was provided to each plant for 14 days, with the modified LA solution configured according to P Dluzniewska et al [3 in LA solution was replaced by 1 mM NaNO3 (S1), 500 μM NH4NO3 (S2) and 1 mM NH4Cl (S3). The nutrient solution was changed every other day. At each treatment level, eighteen plants showing similar growth performance were used for further study.Plantlets of ka et al . Subsequka et al . The plaRoot lengths (length of taproots from the stem end to the root tip) were measured with a ruler. The root tip was compressed with a transparent agent  and observed and measured under a microscope. The location of lateral root initiation was measured under a microscope. The number of lateral roots within a range of 1 cm from the start site of lateral root initiation was counted under a microscope as an indicator of lateral root density. Six plants were measured at each treatment level, and three biological replicates were performed.3, 500 μM NH4NO3 and 1 mM NH4Cl. Each root sample was wrapped in aluminum foil and snap-frozen in liquid nitrogen. A ball mill  was used to grind each root sample into fine powder, which was stored at -80 °C. The same amounts of fine powder were extracted from the root samples of six plants and mixed evenly to form a mixed sample. Thus, three mixed samples were obtained.The whole roots of each plant were carefully cleaned with a modified LA solution containing 1 mM NaNOTotal RNA from the samples was isolated using TruSeq Small RNA Prep Kits  to obtain a small RNA library. Three small RNA libraries for each treatment level were constructed and sequenced on an Illumina HiSeq 2500 sequencer  at LC Science  according to the protocol recommended by the supplier. All of the small RNA sequences are available in the Sequence Read Archive (SRA) under project ID PRJNA631845.Known and novel miRNAs were identified according to the procedure proposed by J Zhou et al . Raw Ill2(fold change) of the differentially expressed miRNAs was determined by the TPM ratio of S1 vs. S2 or S3 vs. S2. P values of less than 0.05 were considered significantly differentially expressed miRNAs. Cytoscape software (3.5.1) was used to construct heatmaps with significantly differentially expressed miRNAs. S1_1, S1_2 and S1_3 represent three biological repeats of S1, S2_1, S2_2 and S2_3 represent three biological repeats of S2, and S3_1, S3_2 and S3_3 represent three biological repeats of S3.The frequency of the miRNAs in each sample was normalized to transcripts per million (TPM) values. The logA total RNA purification kit  Science, Hangzhou, China) was used to extract total RNA from the samples, and a NanoDrop 2000 spectrophotometer  was used to assess the RNA quality according to a 260/280 nm ratio > 2.0. Three independent RNA samples of equal quantities at the same treatment level were mixed to produce one degradation library. Then, three degradome samples  were sequenced on the Illumina HiSeq2500 platform . Degradome library construction was performed according to Xu X et al . Data anTo determine the expression profiles of the target genes, three cDNA libraries from each treatment were constructed from the RNA samples, and transcriptomic sequencing was performed on an Illumina HiSeq2000/2500 sequencer (Illumina) at Lianchuan Science  according to the protocol recommended by the supplier. The transcriptomic sequencing data were also submitted to the SRA under project ID PRJNA631840.P. × canescens for annotation, and the degradome reads were mapped according to the P. × canescens transcriptomic data (http://aspendb.uga.edu/index.php/databases/spta-717-genome). After the miRNA, degradome sequencing and transcriptomic sequencing data were combined as described by M Pertea et al [2(fold change) values of differentially expressed target genes were calculated using the FPKMs of genes determined under S1 vs. S2 or S3 vs. S2. Differentially expressed target genes with P values of less than 0.05 and absolute log2 (fold change) values greater than 1 were considered significantly differentially expressed target genes.For each library, all sequences were processed by filtering out adaptor sequences and low-quality sequences. Subsequently, all clean tags were mapped to unigenes assembled for ea et al , the fraTo clarify the functions of the target genes, a Gene Ontology (GO) functional classification  and KEGGU6 and ACTIN2/7, were selected as reference genes for the validation of miRNAs and target genes, respectively [To verify the significantly differentially expressed miRNAs and their target genes, the samples used for sequencing as described above were employed for qRT–PCR validation using the PrimeScript™ RT reagent Kit and TB Green qRT–PCR kit  as suggested by J Zhou et al . Thirty-ectively .Nicotiana benthamiana leaves according to the methods of J Zhou et al [Agrobacterium tumefaciens strain GV3101 by electroporation. The Agrobacterial cell cultures were inoculated overnight at 28 °C. As previously described [N. benthamiana leaves. After incubation in the dark for two days, the infiltrated leaves were harvested for qRT–PCR. analysis with gene-specific primers , and the different N forms tested were included as a factor. When the P value of the ANOVA F test was less than 0.05, the differences between the means were considered significant.Statistical analysis of the data was performed using Statgraphics software . Before statistical analysis, the data were tested for a normal distribution. All the data were analyzed by Additional file 1. Supporting informationAdditionalsupporting information can be found in the online version of this article: Supplementary Fig.1.Lengths of total small RNAs (a) and unique small RNAs (b) in the S1, S2 and S3libraries.Supplementary Fig.2. Validation of significantly differentially expressed miRNAs. Supplementary Fig. 3.Analysis of GO functional classification of identified differentially expressedtarget genes. S1 vs. S2 (a) and S3 vs. S2 (b). Supplementary Fig. 4. KEGG pathway analysis of identified differentially expressed target genes. S1 vs. S2(a) and S3 vs. S2 (b).Additional  file 2:  Supplementary Table1. Primers used for qRT–PCR.Supplementary Table 2. Distribution of smallRNAs in different categories.Supplementary Table 3.Identified known miRNAs.Supplementary Table 4.Identified miRNAfamilies.SupplementaryTable 5. Identified novel miRNAs.Supplementary Table 6.Significantly differentially expressed known and novelmiRNAs.Supplementary Table 7.Degradome sequencing forthe identification of target genes of known and novel miRNAs.Supplementary Table 8. Target genes ofsignificantly differentially expressed miRNAs.Supplementary Table 9.Annotated analysis of the significantly differentially expressed target genesof DEmiRNAs.


Background: The prevalence of non-alcoholic fatty liver disease (NAFLD) keeps growing recently.Purpose: To investigate the effects and mechanisms of naringenin (NAR) on NAFLD.Methods: High-fat diet (HFD)-induced NAFLD rats were orally administered with NAR at 10, 30, and 90 mg/kg for 2 weeks. The serum level of triglyceride (TG), total cholesterol (TC), glutamic-oxaloacetic transaminase (AST), and glutamic-pyruvic transaminase (ALT) was measured. The hepatic histology was detected by H&E and oil red O staining. L02 and Huh-7 cells were induced by sodium oleate to establish a NAFLD cell model. The effects of NAR on lipid accumulation were detected by oil red O staining. The glucose uptake and ATP content of 3T3-L1 adipocytes and C2C12 myotubes were measured. The expression of proteins of the AMPK signaling pathway in 3T3-L1 adipocytes and C2C12 myotubes was assessed by Western blotting. The mitochondrial biogenesis of 3T3-L1 adipocytes and C2C12 myotubes was measured by mitotracker orange staining and Western blotting. The biomarkers of autophagy were detected by Western blotting and immunofluorescence. The binding of NAR to AMPKγ1 was analyzed by molecular docking. Chloroquine and compound C were employed to block autophagic flux and AMPK, respectively.Results: NAR alleviated HFD-induced NAFLD in rats at 10, 30, and 90 mg/kg. NAR attenuated lipid accumulation in L02 and Huh-7 cells at 0.7, 2.2, 6.7, and 20 μM. NAR increased glucose uptake, decreased the ATP content, activated the CaMKKβ/AMPK/ACC pathway, and enhanced the mitochondrial biogenesis in 3T3-L1 adipocytes and C2C12 myotubes. NAR increased autophagy and promoted the initiation of autophagic flux in 3T3-L1 preadipocytes and C2C12 myoblasts, while it inhibited autophagy in NAFLD rats, 3T3-L1 adipocytes, and C2C12 myotubes. Molecular docking showed that NAR binds to AMPKγ1. Compound C blocked effects of NAR on lipid accumulation and autophagy in L02 cells.Conclusion: NAR alleviates NAFLD by increasing energy expenditure and regulating autophagy via activating AMPK directly and indirectly. The direct binding of NAR and AMPKγ1 needs further validation. Non-alcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease, which global prevalence has reached 24% and keeps rising due to the increasing prevalence of obesity worldwide . NAFLD ide novo lipogenesis and lead to NAFLD  is a key energy sensor and metabolic homeostasis regulation center in mammalian cells . When AMThe most recommended treatment for NAFLD is weight loss by improving lifestyle and dietary . Althougin vivo studies of NAR were conducted in large doses (50–100 mg/kg) and long-term use (4–12 weeks)  . Additioin vitro experiments.We established the high-fat diet (HFD)-induced NAFLD model in rats, and orally administered NAR at 10, 30, and 90 mg/kg for 2 weeks. The effects and mechanisms of NAR on lipid accumulation and energy expenditure were investigated by 15H12O5) with purity above 99% was commercially obtained from Herbest Biotechnology . Rats of the NAR groups were orally administered with NAR daily for 2 weeks. Rats of the ND group and HFD group were orally administered with saline for 2 weeks. Rats continued their diet until being sacrificed. Serum and liver samples were obtained.The experiment was conducted following the Guidance on the Care and Use of Laboratory Animals  and approved by the Medical Ethics Committee of Sichuan University (No. K2020435). Male Sprague-Dawley rats aged 8 weeks were commercially obtained . All together 25 rats were randomly divided into five groups: normal chow diet (ND) group, HFD group, NAR 10 mg/kg group, NAR 30 mg/kg group, and NAR 90 mg/kg group, with five rats per group. Rats of the ND group were fed with an ND  for 12 weeks. Rats of the HFD group and NAR groups were fed with an HFD  for 12 weeks . NAR (C1Serum triglyceride (TG), total cholesterol (TC), glutamic-oxaloacetic transaminase (AST), and glutamic-pyruvic transaminase (ALT) were measured using TG colorimetric assay kit based on glycerol phosphate oxidase-peroxidase method, TC colorimetric assay kit based on cholesterol oxidase-peroxidase method, ALT colorimetric assay kit, AST colorimetric assay kit, respectively  . The absLiver samples of rats were fixed with 4% paraformaldehyde  in phosphate buffer saline (PBS) at 4°C for 16 h. Then samples were dehydrated, embedded in paraffin, sectioned into 6  μm slides, and stained using 0.5% hematoxylin and eosin . All imaL02 cells, 3T3-L1 preadipocytes, and C2C12 myoblasts were obtained from the American Type Culture Collection. Huh-7 cells were a kind gift from Prof. Lang Bai of West China Hospital, Sichuan University. L02 cells were cultured with Roswell Park Memorial Institute (RPMI) 1640 medium  supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Huh-7 cells were cultured with Dulbecco’s Modified Eagle medium  supplemented with 10% FBS and 1% penicillin/streptomycin.To differentiate into insulin resistant 3T3-L1 adipocytes, 3T3-L1 preadipocytes were induced with the DMEM supplemented with 10% NBCS, 1 µM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 1 μg/ml insulin for 48 h. Then the medium was changed to the DMEM supplemented with 10% NBCS and 1 μg/ml insulin for 48 h. Then the medium was changed to the DMEM supplemented with 10% NBCS and 1% penicillin/streptomycin. More than 90% of 3T3-L1 preadipocytes differentiated to adipocytes after induction for 8–10 days, which showed a fat cellular phenotype filled with lipid droplets.2.C2C12 myoblasts were cultured in the DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin. C2C12 myoblasts were treated with DMEM medium supplemented with 2% horse serum  for 48 h to induce differentiation into insulin resistant C2C12 myotubes. Mononucleated C2C12 myoblasts fused to form multinucleated C2C12 myotubes after being induced for 4–6 days. All cells were maintained at 37°C in a humidified atmosphere containing 5% COFresh liver samples were embedded in optimal cutting temperature compound (O.C.T.) and dissected into 10 μM sections. Then sections were stained with oil red O solution (3 mg/ml) and rinsed as described . L02 and3T3-L1 adipocytes and C2C12 myotubes were treated with metformin  as the positive control, phloretin  as the negative control, and NAR for 4 h. Glucose uptake was measured using the 2-[N- amino]-2-deoxy-glucose (2-NBDG) glucose uptake assay kit  . Images 3T3-L1 adipocytes and C2C12 myotubes were treated with NAR for 24 h. Total ATP content was measured using an ATP colorimetric assay kit  . The absAs for rat livers, 100 mg freshly frozen samples were cut into small pieces, lysed using 400 μl tissue lysate including 4 μl phenylmethylsulfonyl fluoride, and ground with homogenizer on ice. Then samples were centrifuged at 10,000 rpm for 5 min at 4°C. The supernatant was collected. All together 50 μl 5 × SDS-PAGE sample loading buffer was added to the supernatant and heated at 98°C for 20 min. As for cellular samples, cells were washed twice with PBS, harvested, and centrifuged at 1,500 rpm for 2 min at 4°C. Then samples were resuspended in the 1 × SDS-PAGE sample loading buffer, and heated at 98°C for 10 min. Proteins were separated on a 10% SDS-polyacrylamide gel and transferred to a polyvinylidene fluoride membrane. Transferred membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween 20 and then incubated with the primary antibody at RT for 2 h, including p-AMPK , AMPKα , p-ACC , AAC  CaMKKβ , LKB1 , p-LKB1 , PGC1α , NRF1 , TFAM , SIRT1 , p-ULK1 , ULK1 , LC3 , p62 , and GAPDH . Then membranes were washed with Tris-buffered saline with 0.1% Tween 20 and incubated with horseradish peroxidase-conjugated secondary antibody  at RT for 1 h . SignalsCells were seeded on coverslips in 24-well plates and treated with NAR as indicated. Then cells were fixed with 4% PFA for 20 min and permeabilized with 0.5% Triton X-100 for 15 min. Cells were blocked with 4% bovine serum albumin for 1 h, cultured with the primary antibody against LC3  at RT for 2 h, and incubated with secondary antibody conjugated to fluorescein isothiocyanate  at RT for 1 h . The nucCells were seeded on coverslips in 24-well plates and treated with NAR as indicated. Cells were stained with 250 nM mitotracker orange probe staining solution  for 30 min . Then cehttp://www.rcsb.org/) and used as the receptor structure for molecular docking. 3D structure of NAR  was obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/), converted to MOL2 format with PyMOL 2.4 software , and used as ligand structure for molecular docking. GetBox Plugin, a plugin of PyMOL 2.4 software, was used to obtain the active site for molecular docking. LeDock 1.0 software  was used for molecular docking  subunit of human AMPK in complex with AMP  was downloaded from the RCSB protein data bank . p values less than 0.05 were considered statistically significant.All experiments were performed in triplicate and data were expressed as the mean ± SD. All data were analyzed with the Student’s Serum levels of TG, TC, ALT, and AST of HFD-induced NAFLD rats were dose-dependently decreased by NAR at 10, 30, and 90 mg/kg . As deteL02 and Huh-7 cells were induced by sodium oleate to establish the NAFLD cell model. As detected by oil red O staining, NAR significantly inhibited lipid accumulation in L02  and Huh-As detected by Western blotting, NAR increased the expression of CaMKKβ, promoted the phosphorylation of AMPK and ACC in 3T3-L1 adipocytes  and C2C1NAR dose-dependently increased endogenous LC3 puncta in both cells . NAR pro). NAR increased the expression of p62 in 3T3-L1 adipocytes , an AMPK inhibitor, was employed. NAR inhibited lipid accumulation in sodium oleate-treated L02 cells, which was reversed by Comp C . NAR enh0–48h for quercetin and hesperetin were similar, whereas the AUC0–48h of NAR was higher. Thus, the relative bioavailability of NAR was higher than quercetin and hesperetin. Most studies of NAR’s effects for NAFLD on animal models were conducted in large doses (50–100 mg/kg) and (or) in a long time (4–12 weeks)  or phenolic compounds have multiple pharmacological activities such as regulating glucose metabolism , anti-in2 weeks) . Wang et2 weeks) . We oral2 weeks) . NAR was2 weeks) . These rin vitro experiments. NAR did increase the glucose uptake and decrease the ATP content of 3T3-L1 adipocytes and C2C12 myotubes. NAR in the aglycone of naringin, that, in turn, is reported to increase AMPK phosphorylation  in the liver of diabetic mice . NAR is As a famous energy sensor and regulator, AMPK plays a key role in energy expenditure . InhibitNAR was reported to reduce the activity of SIRT1 in cell-free histone deacetylase activity assay. NAR increased the expression and activity of SIRT1 in lipopolysaccharide-induced-THP-1 cells. These reports suggested that NAR may have a dual role in activating SIRT1. Our results showed that NAR did not increase the expression of SIRT1 at 0.7–6.7 μM, while the reason remains unclear. NAR binds to estrogen receptors (ER) α and β , and actRoles of autophagy in NAFLD remain controversial. Drugs against NAFLD by either increasing autophagy or inhibiting autophagy were reported . We obseOur study has a couple of limitations. The binding of NAR and AMPKγ1 was explored by molecular docking and Comp C, which cannot demonstrate the direct binding of NAR and AMPKγ1. NAR regulated autophagy according to the cellular status whose mechanisms need further clarification.In conclusion, we demonstrated that NAR attenuates NAFLD in short-term use of low doses by enhancing energy expenditure, which may be a candidate for NAFLD, even metabolic disorders such as obesity. The mechanisms involve regulating autophagy by directly or indirectly activating AMPK.


TP53 gene, a P2 transcription initiation site is located and this transcript encodes two p53 isoforms: Δ133p53 and Δ160p53. Here, the secondary structure of the 5′-terminal region of P2-initiated mRNA was characterized by means of the SHAPE and Pb2+-induced cleavage methods and for the first time, a secondary structure model of this region was proposed. Surprisingly, only Δ133p53 isoform was synthetized in vitro from the P2-initiated p53 mRNA while translation from both initiation codons occurred after the transfection of vector-encoded model mRNA to HCT116 cells. Interestingly, translation performed in the presence of the cap analogue suggested that the cap-independent process contributes to the translation of P2-initiated p53 mRNA. Subsequently, several antisense oligonucleotides targeting the 5′-terminal region of P2-initiated p53 mRNA were designed. The selected oligomers were applied in in vitro translation assays as well as in cell lines and their impact on the Δ133p53 synthesis and on cell viability was investigated. The results show that these oligomers are attractive tools in the modulation of the translation of P2-initiated p53 mRNA through attacking the 5′ terminus of the transcript. Since cell proliferation is also reduced by antisense oligomers that lower the level of Δ133p53, this demonstrates an involvement of this isoform in tumorigenesis.The p53 protein is expressed as at least twelve protein isoforms. Within intron 4 of the human  TP53 gene, a P2 transcription initiation site is located. This transcript encodes two p53 isoforms: Δ133p53 and Δ160p53. In addition to the Δ133p53 isoform, also referred to as Δ133p53α, isoforms truncated from the carboxyl terminus, Δ133p53β and Δ133p53γ, have also been defined. Analogously to Δ133p53 isoforms, Δ160p53α, Δ160p53β, and presumably also Δ160p53γ variants are generated in the cell -labelled DNA primers:Prior to the PbAATCAACCCACAGCTGCACAGGGCAG-3′,R133p53UTR2: 5′-GTGAACAGATAAAGCAACTGG-3′,R133p53UTR3: 5′-TTTGAGATAGGGTCTTGCTCTGTCAC-3′,R133p53UTR6: 5′-CAACCTCCGTCATGTGCTGTGAC-3′,R160p53UTR: 5′-ACAGGGCAGGTCTTGGC-3′.R133p53UTR2_2: 5′-were first denatured for 5 min at 65°C. The reaction mixture contained: 1X FS buffer, 200 U SuperScript IV Reverse Transcriptase (Invitrogen), 0.5 mM dNTPs Mix, and 10 U RiboLockRNase inhibitor (Thermo Fisher Scientific). The reaction was conducted for 10 min at 55°C followed by 15 min at 70°C. The sequencing reaction was prepared in the same conditions as described above, using RNA samples and 0.2 mM dideoxy-terminating nucleotides. Finally, the solution containing 8 M urea, 20 mM EDTA and xylene cyanol dye was added to the samples, incubated for 3 min at 95°C and loaded on 8 M urea, 8% polyacrylamide gels. After electrophoresis the gels were scanned by phosphorimaging using the FLA 5100 image analyzer (FujiFilm).2, 130 mM KCl, and 0.1 mM EDTA was added to sample and incubated at 37°C for 10 min. In this buffer a modification reaction was performed using NMIA dissolved in DMSO . The control reaction contained DMSO without NMIA. Both modification and control reactions were incubated at 37°C for 50 min and the RNA was precipitated and resuspended in water. An analysis of the modification sites was performed by the primer extension reaction, in the same manner as described above for Pb2+-induced RNA cleavage.0.4 μM of RNA was denatured in a buffer containing 10 mM Tris pH 8.0, 100 mM KCl, and 0.1 mM EDTA, at 90°C for 3 min and cooled (0.1°C/s) to 4°C. Next, buffer: 40 mM Tris pH 8.0, 5 mM MgClRNAstructure 5.6 program.For the purpose of SHAPE data analysis , followiIn vitro translation was performed using Rabbit Reticulocyte Lysate (RRL) System (Promega). The reaction contained 8.75 μl RRL, 20 μM aminoacid mixture minus methionine, 0.5 μl 35S-methionine , 10 U of RiboLockRNase inhibitor (Thermo Fisher Scientific) and 1.25 pmol of capped RNA, which was previously denatured for 5 min at 65°C. The final volume of the reaction was 12.5 μl and it was conducted for 90 min at 30°C. Afterwards, the reaction was treated with 0.16 μg of RNase A for 5 min at 20°C, and denatured for 2 min at 80°C in the presence of SDS Sample Buffer and 100 mM DTT. The reaction products were analyzed in 15% SDS-PAGE, followed by radioisotope imaging with FLA 5100 image analyzer (FujiFilm).7GpppG cap analog from 5 to 750 μM (Epicenter Biotechnologies) and the same concentrations of magnesium acetate, for 15 min at 30°C. Afterwards, 20 μM amino acid mixture minus methionine, 0.5 μl 35S-methionine , 10 U of RiboLockRNase inhibitor (Thermo Fisher Scientific) and 1.25 pmol of previously denatured RNA were added to the RRL samples and incubated for 90 min at 30°C. The samples were treated with RNase A and analyzed in 15% SDS-PAGE as described above.For translation inhibition assay, RRL was pre-incubated with an increasing concentration of mE. coli RNase H (Thermo Fisher Scientific), 1 pmol of RNA was denatured in a buffer containing: 40 mM Tris-HCl pH 8.0, 40 mM KCl, 10 mM MgCl2, 1 mM DTT, 0.1 mM EDTA for 5 min at 70°C and then for 5 min at 37°C. Then, 5 pmol of each antisense oligonucleotide was added and incubated for 5 min at 37°C. The cleavage reactions were induced by adding 0.5 U of RNase H enzyme. The reaction mixtures were incubated for 7.5 min at 37°C. The reactions were stopped by adding an equal volume of 20 mM EDTA. The reaction products were analyzed by 1% agarose gel in 1xTBE.Prior to digestion with OMe PS antisense oligonucleotides at a final concentration between 0.075 and 0.5 μM were transfected into cells using Lipofectamine RNAiMax reagent, according to the manufacturer’s transfection protocol (Invitrogen) [MCF-7, HepG2 and H1299 cells were purchased from ECACC. MCF-7 cells were maintained in DMEM, HepG2 in MEM and H1299 cells were cultured in McCoy’s medium. All media (Sigma) were supplemented with 10% fetal bovine serum, non-essential amino acids (Gibco-BRL), 100 U/ml of penicillin G, 0.1 mg/ml of streptomycin sulphate (Sigma). The plasmid transfection in the final concentration of 1 ng/μl was performed using Lipofectamine 3000 (Thermo Fisher Scientific) or Dharmafect 2 (Dharmacon) transfection reagents. After 4 hours 2′-itrogen) . Cells wFor Western blots, cell lysates were prepared in a buffer: 62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT and protease inhibitor (Roche). Total cell lysates were incubated for 5 min at 95°C and then loaded on a 15% SDS-PAGE gel and proteins were transferred to a PVDF membrane . The blot was probed with mouse DYKDDDDK Tag Polyclonal Antibody (FLAG) (Thermo Fisher Scientific), rabbit anti-p53 monoclonal antibody Pab421 (Abcam), rabbit β-Actin Monoclonal Antibody , mouse GAPDH Monoclonal Antibody (Santa Cruz Biotechnology). Primary antibody was detected by Goat Anti-Mouse-HRP or Goat Anti-Rabbit-HRP (Thermo Fischer Scientific) and visualized using the enhanced chemiluminescent visualization (ECL) system (Thermo Fisher Scientific Pierce).Flow cytometry measurements were performed after 24, 48 and 72 hours of oligomer transfection using a LIVE/DEAD Viability/Cytotoxicity two-color flow cytometry assay (Thermo Fisher Scientific), following the manufacturer’s protocols. The procedure was optimized in the Laboratory of Subcellular Structures Analyses of our Institute . BrieflyTP53 gene, covers the region from intron 1 to the proximal part of exon 5, including about 1500 base pairs [Initially, it has been thought that the promoter of P2-initiated mRNA, found in the se pairs . Furtherse pairs . The resse pairs ,6.in vitro transcription. The transcript starts in intron 4 of the TP53 gene and includes 250 final nucleotides of the intron 4 sequence. In Δ133p53 mRNA, the AUG3 initiation codon for Δ133p53 protein is located at nucleotide positions 279–281, while AUG4 codon for Δ160p53 isoform is located at positions 360–362 (2+-induced cleavage [RNAstructure 5.6 program [2+-induced cleavage data was chosen.The model P2-initiated p53 mRNA transcript Δ133p53 mRNA) was synthesized by  360–362 . The strcleavage ,32 methop53 mRNA 2+-induced cleavages  with radiolabeled methionine  and mRNA(AUG3CUA4), in which the first or the second AUG codon was changed to a CUA triplet coding leucine. Unexpectedly, translation of mRNA(CUA3AUG4) in RRL yielded two protein products of comparable intensity  was used as the control. Out of the three oligomers tested, the lowest protein level was observed in the presence of oligomer No. 18, which was bound to the mRNA with the highest affinity among the tested oligonucleotides. An approximately 40% decrease of translation efficiency at 0.15 μM concentration of oligomer No. 18 was observed in comparison to the reaction with the control oligomer while at the 0.075 μM concentration of oligomer No. 18, a 20% decrease of translation occurred. Oligonucleotides No. 3 and 4 also caused reduction of the Δ133p53 protein synthesis, yet to a smaller extent compared with oligomer No. 18 . Analysis of fluorescence intensity indicated the number of live and dead cells for each experimental condition. Surprisingly, for these fast growing cells we observed changes in cell proliferation  already in vitro or it is an exclusive initiation site, whereas translation from AUG4 proceeded with low efficiency only when AUG3 codon was mutated. However, when HCT116 cells were transfected with Δ133p53-FLAG mRNA two protein products, originated from both AUG3 and AUG4 codons were observed although translation from AUG4 occurred with much lower efficiency  . The 5′ ficiency .in vitro and in cellulo conditions in the ratio of approx. 5:1 [in vitro occurring only from AUG3 and much weakly from AUG4 than from AUG3 in HCT116 cells transfected with vector-encoded Δ133p53-FLAG mRNA contradicts the expectations based on the Kozak’s rule on the preferred nucleotide context of translation initiation codons [AUGUCUCAACAAG differs substantially from the Kozak’s consensus context of AUGGGCCGCC(A/G)CC and AUG4 is embedded in the nucleotide context of AUGGGUCCGCGCC, which much better corresponds to that optimal for efficient translation initiation  show either no signs or low levels of Δ160p53 expression [The Δ160p53 isoform, the second protein product which is synthesized from Δ133p53 mRNA  has beenpression . Thus, oTP53 it seems likely that it is the activation of p53 that trans-activates the Δ133p53 isoform [TP53 MCF-7 breast cancer cells that express all WT p53 isoforms except Δ133p53β, enhances the invasive activity of MCF-7 [TP53 mutation in glioblastoma that aids tumor progression by promoting an immunosuppressive and chemo-resistant environment [So far, not all functions of the Δ133p53 and Δ160p53 isoforms have been defined and regulation of their expression is still uncovered. As elevated Δ133p53 is only observed in cells with WT  isoform ,39. It i isoform . A metab isoform . Interesof MCF-7 . A stronironment . Moreoveironment . An incrironment . Interesironment .We applied antisense oligonucleotides targeting the 5′-terminal region of Δ133p53 mRNA aiming to inhibit translation initiation from this mRNA. It is known that accessibility to oligonucleotide hybridization does not reflect the RNA secondary structure in a simple, straightforward manner. Therefore, a comprehensive analysis of the sites accessible to oligomer hybridization was performed using the RNase H approach, based on which several antisense compounds were designed and subsequently tested in RRL translation system Figs  and 5. TSeveral antisense oligomers and siRNAs have earlier been reported to attack the 5′-terminal region of Δ133p53 mRNA. However, almost all oligonucleotide tools have targeted only two sequence stretches of that region. The first targeted sequence spans nucleotides at positions G149 and U173 which form an unstable hairpin G152-C187 in the secondary structure model of that region . In our Bax and downregulation of Bcl-2 [Within the first G149-U173 region, siRNA has been targeted, which decreased endogenous Δ133p53α in U2OS cells, coupled to doxorubicin treatment [of Bcl-2 . A relatof Bcl-2 . Within of Bcl-2 . In humaof Bcl-2 . HFK andof Bcl-2 . Silenciof Bcl-2 . FinallyWe also determined cell line viability upon transfection of the tested antisense oligomers . We chosS1 File(PDF)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


Cellular homeostasis depends on robust protein quality control (PQC) pathways that discern misfolded proteins from functional ones in the cell. One major branch of PQC involves the controlled degradation of misfolded proteins by the ubiquitin-proteasome system. Here ubiquitin ligases must recognize and bind to misfolded proteins with sufficient energy to form a complex and with an adequate half-life to achieve poly-ubiquitin chain formation, the signal for protein degradation, prior to its dissociation from the ligase. It is not well understood how PQC ubiquitin ligases accomplish these tasks. Employing a fully reconstituted enzyme and substrate system to perform quantitative biochemical experiments, we demonstrate that the yeast PQC ubiquitin ligase San1 contains multiple substrate binding sites along its polypeptide chain that appear to display specificity for unique misfolded proteins. The results are consistent with a model where these substrate binding sites enable San1 to bind to misfolded substrates avidly, resulting in high affinity ubiquitin ligase-substrate complexes. All living cells are burdened with the task of disposing of misfolded or damaged proteins . Termed Addressing how ubiquitin ligases bind to and recognize misfolded proteins requires robust in vitro reconstituted systems that enable biochemical, biophysical, and structural biological inquiries into function. This is challenging work ,23,24 owSaccharomyces cerevisiae enzyme that recognizes and ubiquitylates misfolded proteins in the nucleus [Amongst the best characterized PQC-specific ubiquitin ligases is San1, a  nucleus ,45,46,47 nucleus . Unlike Saccharomyces cerevisiae nuclear PQC ubiquitin ligase San1 was shown to contain multiple contiguous disordered regions in the primary structure that appear to bind to San1 protein substrates [In a landmark study, the bstrates , leading1–303 was purified similarly with a few notable modifications. Briefly, proteins were expressed in Escherichia coli using Rosetta 2(DE3)pLysS competent cells . Bacterial cells were grown at 37 °C to an optical density of 0.8–1.0, after which expression was induced with 0.1 mM IPTG for 2 h followed by centrifugation and storage of the cell pellets at −80 °C. Bacterial cell pellets were solubilized in lysis buffer containing 30 mM Tris, pH 7.5, 200 mM NaCl, 5 mM DTT, 1 mM EDTA, 10% glycerol, and protease inhibitor cocktail  and disrupted by multiple rounds of sonication. Lysates were prepared by centrifugation and then incubated with Glutathione Sepharose 4B beads  for 3 h at 4 °C. Beads were then collected and washed repeatedly with lysis buffer lacking protease inhibitors and EDTA. Recombinant GST- San11–303 protein was eluted in a buffer containing 50 mM tris, pH 8.0, 200 mM NaCl, and 40 mM glutathione. The protein was then incubated with TEV protease overnight at 4 °C, followed by loading onto a 1 mL HisTrap HP column  that had been equilibrated in buffer A containing 50 mM HEPES, pH 7.5, 200 mM NaCl, 20 mM imidazole, and 5% glycerol. Histidine-tagged San1 proteins were eluted from the column using a linear gradient of buffer B containing 50 mM HEPES, pH 7.5, 200 mM NaCl, 300 mM imidazole, and 5% glycerol. Fractions containing San11–303 were concentrated and then injected onto a Superdex 200 gel filtration column . Fractions containing San11–303 were concentrated  to approximately 10 µM and flash frozen in liquid nitrogen prior to storage at −80 °C.Since wild-type San1 protein has been shown to rapidly auto-ubiquitylate, all experiments performed for these studies were with San1 constructs where lysine residues had been changed to arginines. Full-length San1 was purified as previously described . San11–3San1 peptide  N-CGSRRGSYNASSGEQMLSRTGFFLVLIVGQPLHNPVK-CtermKR San1 peptide N-CGSRRGSYNASSGEQMLSRTGFFLVLIVGQPLHNPVR-CtermHuman E1 and Ubc1p were purified as previously described . Recombi2, and 2 mM DTT. All reactions contained 0.25 µM radiolabeled full-length San1 or San11–303 and were supplemented with 0.1% tween-20. Reactions were then incubated at room temperature in either the absence or presence of 5 µM San1 peptide for two minutes followed by the addition of a 1:100 molar ratio of chymotrypsin . Time-points were quenched in 2X SDS-PAGE and boiled for 5 min at 95 °C. Substrate and products were resolved by SDS-PAGE on 4–20% gels, dried, and exposed on a phosphor screen. Autoradiography was performed using a Typhoon 9410 imager. Quantification of substrate and products were performed with ImageQuant software . San1-trypsin digestion assays were performed similarly except in a buffer containing 30 mM Tris, pH 7.5, 5 mm MgCl2, 2 mM ATP, 2 mM DTT, and 0.1% Tween-20 and with a 1:20 molar ratio trypsin to San1. Reactions with Firefly Luciferase  and trypsin were performed similarly as above except all steps were performed at 42 °C prior to quenching.San1 chymotrypsin digestion assays were performed in a buffer containing 30 mM Tris, pH 7.5, 100 mM CaCl32P labeled ATP  and cAMP-dependent Protein Kinase  for 1 h at 30 °C in a reaction buffer that had been supplemented with tween-20 (0.1%). All reactions were performed in a buffer containing 30 mM Tris, pH 7.5, 5 mm MgCl2, 2 mM ATP, 2 mM DTT, and 0.1% Tween-20. Human E1 (1 µM), WT ubiquitin (60 µM), Ubc1 (10 µM), and either full-length San1 or San11–303 (0.5 µM) were sequentially added to Eppendorf tubes and incubated for 2 min at room temperature. Next, 3 µM radiolabeled San1 peptide, 3 µM radiolabeled San1 peptide mixed with 3 µM unlabeled San1 peptide, or 3 µM radiolabeled San1 peptide mixed with 6 µM unlabeled San1 peptide were then added to initiate the respective ubiquitylation reactions. Reactions were quenched at various time-points in 2X SDS-PAGE buffer and substrate and ubiquitylated products were separated by SDS-PAGE on 4–20% gels . Gels were dried and exposed to phosphor screens for autoradiography. The quantification of substrates and products was performed as described in the limited proteolysis section. The fraction of ubiquitylated San1 peptide was calculated by dividing the amount of peptide that had been modified by one or more ubiquitins by the total signal in the lane.The San1 peptide was radiolabeled (50 µM) in the presence of γ-2, 2 mM ATP, 2 mM DTT, and 0.1% Tween-20. E1 (1 µM), WT human Ub (60 µM), and Ubc1 (10 µM) were incubated at room temperature to form activated ubiquitin-Ubc1 complex (tube 1). In a separate tube, full-length San1 or San11–303 (1 µM) and labeled San1 Peptide (1 µM) were incubated to form a complex (tube 2). Ubiquitylation reactions were initiated by mixing tubes 1 and 2 together at room temperature. KR San1 peptide (10 µM) was added to either tube 1 or tube 2 as a negative control or for single-encounter ubiquitylation, respectively. Substrate and products were separated by SDS-PAGE on 4–20% gels, followed by processing and quantification as described in the multi-turnover ubiquitylation reactions section.All single-encounter reactions were performed in a buffer containing 30 mM Tris, pH 7.5, 5 mm MgCl1–303 for 5 min at room temperature. Binding reactions were diluted with 1 mL of nickel wash buffer containing 30 mM Tris, pH 7.5, 250 mM NaCl, 20 mM Imidazole, 0.1% Tween-20, and 5% Glycerol and incubated with 20 µL Nickel-NTA Agarose beads  with gentle agitation for 1 h at room temperature. Reactions were then spun down at 1000× g for 2 min and 1 mL of additional wash buffer was introduced to the beads in the absence or presence of 5 µM unlabeled San1 peptide. Reactions were incubated for an additional 2 h under gentle agitation at room temperature. Beads were spun down and washed twice with wash buffer (containing no cold peptide). In total, 20 µL of 2X SDS Page buffer was added to the beads and boiled for 5 min at 95 °C. Bead-bound proteins were resolved by SDS-PAGE on 4–20% gels, dried, and exposed to a phosphor screen to perform autoradiography. The fraction of radiolabeled substrate bound to the beads was calculated as a fraction of the total input amount.For binding reactions containing peptide substrate, the San1 peptide was radiolabeled (50 µM) as described in the multi-turnover ubiquitylation reactions section. A total of 5 µM Radiolabeled San1 Peptide was then incubated with 0.1% tween and either 0.5 µM full-length San1 or KR San11–303 for 5 min at 50 °C. Reactions were diluted with 1 mL of warmed nickel wash buffer and incubated with 20 µL Nickel-NTA Agarose beads with gentle agitation for 1 h at 50 °C. Reactions were then centrifuged at 3000× g for 30 s and washed three times with warmed nickel wash buffer. 20 µL of 2X SDS Page buffer was added to the beads and boiled for 5 min at 95 °C. Bead-bound products were transferred to nitrocellulose paper using a BioRad Semidry Transfer Cell Trans Blot SD and blocked in 5% nonfat milk in TBST for 1 h at room temperature. The membrane was then incubated with anti-luciferase antibody  in 0.5% milk using a 1:5000 dilution overnight at 4 °C. The secondary antibody that had been conjugated to Alexafluor 488  was diluted 1:5000 in 0.1% milk and incubated with the membrane for 1 h at room temperature. Signal was detected using a Typhoon 9410 imager.For binding reactions containing Firefly Luciferase , 0.5 µM luciferase was incubated with either 0.5 µM full-length San1 or KR San12, 2 mM ATP, 2 mM DTT, and 0.1% Tween-20. E1 (1 µM), WT human Ub (60 µM), Ubc1 (10 µM), and either full-length or San11–303 (0.5 µM) were incubated at room-temperature. In competition reactions, unlabeled KR San1 Peptide (10 µM) was added to the mixture and incubated for 2 min at 42 °C. Luciferase (0.5 µM) was then added to initiate the reactions that were then quenched with 2X SDS-PAGE loading buffer at the indicated time points. Substrate and product were resolved by SDS-PAGE on 4–20% gels. Substrates and products were transferred to nitrocellulose paper using a BioRad Semidry Transfer Cell Trans Blot SD and blocked in 5% milk in TBST buffer for 1 h at room temperature. The membrane was next incubated with anti-luciferase antibody  in 0.5% milk and TBST buffer at a 1:5000 dilution overnight at 4 °C. Secondary anti-rabbit antibody  diluted 1:5000 in 0.1% milk was incubated with the membrane for 1 h at room temperature. The membrane was imaged using Western Bright ECL  on a BioRad ChemiDoc XRS+.Reactions were performed in a buffer containing 30 mM Tris, pH 7.5, 5 mm MgCl1–303 hereafter, migrated similarly by SDS-PAGE as the major degradation product of full-length San1 as expected. We reasoned that San11–303 may retain similar levels of activity as full-length if San1 substrate binding sites are redundant.We began our investigation by attempting to improve the reconstituted ubiquitylation system since full-length recombinant San1 protein is highly prone to proteolysis, resulting in degradation products occurring even after multiple rounds of purification and with affinity-based tags at both the N- and C-terminus A,B. Inte1–303 suggested that it may be more amenable to in vitro biochemistry than full-length San1. First, the expression level of San11–303 in Escherichia coli was far higher in comparison with full-length San1, and the final level of purity was also significantly improved . Full-length San1 or San1 peptide . Indeed, peptide , support1–303, a C-terminal truncation that enabled far greater levels of purity in comparison with full-length San1, and encouragingly led to nearly identical results as with full-length. The results are all consistent with a model where San1 binds to misfolded substrates through the action of multiple binding regions that have distinct affinities for unique substrates.It had been known for some time that San1 contains multiple disordered regions, and their systematic deletion in yeast led to defects in both substrate binding and degradation. Our goal was to characterize San1 substrate binding in vitro using direct experimental approaches including biochemical and enzymological assays. While experiments were performed with full-length San1, the presence of several degradation products in that sample made unambiguous interpretation of the results challenging. As such, the same experiments were also performed with San11–303 over a very broad range of substrate concentrations  for full-length San1 or for San11–303. The model also suggests why San11–303 converts a lower fraction of substrate to product than full-length. The truncation of the C-terminus to the RING domain seems to have deleted at least some of the binding sites for the peptide substrate, although alternative explanations cannot be ruled out since the peptide solubility is insufficient to achieve saturation of either San1 protein.An intriguing observation from the kinetic experiments is that the fraction of peptide substrate converted to ubiquitylated product was consistent for both full-length San1 and San1trations . Indeed,Our model also explains why San1 appears to have such remarkable affinity for substrates. The existence of multiple substrate binding sites along a disordered polypeptide chain is likely to result in their achieving proximity with each other. The random coalescing of these binding sites is conceptually similar to the principle of how other avidity-dependent binding events such as affinity chromatography occur. San1 shows avid binding to substrate since the dissociation of a substrate from San1 would likely result in rapid rebinding owing to the high local concentration of unoccupied binding sites. Additionally, similar to affinity chromatography, one means for disrupting the San1-substrate complex is through the addition of excess substrate which was observed during the nickel pull-down binding studies reported here.Our results stand in striking contrast with how more typical ubiquitin ligases create binding energy with their substrates. For instance, the cullin-RING ligases are the largest and perhaps best characterized family of ubiquitin ligases to date . Here, s1–303 is possibly amenable to sophisticated in vitro experiments will lead to more quantitative assays as well as perhaps structural biological efforts, as we consider these prerequisite before a detailed molecular understanding of PQC ubiquitylation can be achieved.Like all investigations, our results point to additional experiments that are necessary to further unravel the mechanism of how PQC ubiquitin ligases recognize and bind to misfolded protein substrates. Nevertheless, the results demonstrate how a PQC-specific ubiquitin ligase has evolved an apparently unique mechanism in comparison with more typical ligases for establishing tight substrate binding, and we sincerely hope that these results will embolden other researchers in the field to further develop quantitative in vitro assays for San1 as well as for their PQC-specific ubiquitin ligases of interest. It is also our hope that the demonstration that San1


The dynamic stability of 2Dwrinkle like-PdQ2 is proven by positive phonon frequencies in the phonon dispersion curve. The optimized structural parameters of wrinkled pentagonal PdQ2 are in good agreement with the available experimental results. The ultimate tensile strength (UTHS) was calculated and found that, penta-PdS2 monolayer can withstand up to 16% (18%) strain along x (y) direction with 3.44 GPa (3.43 GPa). While, penta-PdSe2 monolayer can withstand up to 17% (19%) strain along x (y) dirrection with 3.46 GPa (3.40 GPa). It is found that, the penta-PdQ2 monolayers has the semiconducting behavior with indirect band gap of 0.94 and 1.26 eV for 2D-PdS2 and 2D-PdSe2, respectively. More interestingly, at room temperacture, the hole mobilty (electron mobility) obtained for 2D-PdS2 and PdSe2 are 67.43 (258.06) cm2 V−1 s−1 and 1518.81 (442.49) cm2 V−1 s−1, respectively. In addition, I-V characteristics of PdSe2 monolayer show strong negative differential conductance (NDC) region near the 3.57 V. The Shockly-Queisser (SQ) effeciency prameters of PdQ2 monolayers are also explored and the highest SQ efficeinciy obtained for PdS2 is 33.93% at −5% strain and for PdSe2 is 33.94% at −2% strain. The penta-PdQ2 exhibits high optical absorption intensity in the UV region, up to 4.04 × 105 (for PdS2) and 5.28 × 105 (for PdSe2), which is suitable for applications in optoelectronic devices. Thus, the ultrathin PdQ2 monolayers could be potential material for next-generation solar-cell applications and high performance nanodevices.We studied the physical, electronic transport and optical properties of a unique pentagonal PdQ Among the 2D materials, if targeting application in nanodevices, MX2  type TMDs are technologically favourable materials9. Nowadays, an endless research is going on traditional TMDs layered materials, which is in demand of high mobility, high stability in air and strain modulated bandgap. Of these, many of TMDs has been already synthesized via the various method of exfoliation14. Interestingly, palladium based dichalcogenides has brought 2D with buckled or puckered pentagonal structures under the spotlight because of its unique lattice arrangement and fascinating properties such as fine tuning their physical, elctronic, thermal and optical properties.Atomically thin two-dimensional (2D) structures like, honeycomb lattice graphene2/PtS2 and concluded that noble metals (Pd and Pt) would be a layered structure with sulphur atoms16. Afterwards, in 2015 Wang et al., theoretically reported that 2D PdS2 is semiconductor with moderate indirect band gap up to ~ 1.60 eV, which is very significant in fabrication of nanodevices17. Moreover, Cheng et al.,18 have developed ultrafast Yb doped fiber laser using few layers of PdS2 and indicated that layered PdS2 can be a favourable candidate for photonics application. Some work have been reported on the layered PdSe2 via method of mechanical exfoliated, atomic-resolution scanning transmission electron microscopy (AR-STEM) and chemical vapor deposition (CVD)24. For Example, Akinola et al.,24 had fabricated few layers puckered pentagonal PdSe2 by AR-STEM method and demonstrated that, PdSe2 exhibits good ambipolar semiconducting nature with room temperature electron-apparent field-effect mobility (up to ~ 158 cm2 V−1 s−1) and its stability is remain same up to 60 days. It has been noted that having high mobility (electron/hole) and band gap between 1.2 ~ 1.9 eV of TMD, it makes them suitable for the FETs (field effect transistors) and CMOS applications, respectively28.Gronvold et al., studied the PdS29 had successfully synthesized 2D PdSe2 nanosheets on 300 nm SiO2/Si substract and claimed that it is really promising material to make infrared photodetector due to its high-photoresponsivity (~ 660 A.W−1 under 914 nm laser) in harsh condition, which may be benefitted in military field for night-time detection because of its ability to work at night as well as in bad whether conditions. Beside these, few other reports theoretical are also available on the 2D pentagonal type PdQ2 monolayers34. Deng et al.,31 investigated the strain applied mechanical, electronic and optical properties of PdS2, PdP2 and PtSe2. Except this, none of works have been reported on strain applied mechanical, electronic, optical and electronic transport properties of 2D penta-PdQ2 . Further, the lower band gap of 2D-PdQ2 monolayers has also motivated us to investigate absorbance of solar radiation on nanosheet. Hence, we also studied the strain dependent solar cell power conversion efficiency % (PCE) of penta-PdQ2  monolayers.Recently, Weiting and their research group2 using density functional theory (DFT). After the optimization of structures, we analysed the lattice parameters (Å), band gap (eV) and effective mass (m*). Then after we applied biaxial strain ɛ (%) and obtained its bulk modulus B (GPa). The ultimate tensile strength (UTHS) is also found under the tensile strain (+ ɛ%) for PdQ2 . We also studied the influence of loading on the band edges and obtained carrier mobility (µ2D) along biaxial strain ɛ (%). We have adopted Shockley-Queisser (SQ) method to study Solar cell efficiency η (%) and calculated the maximum power density  monolayers are also reported in this paper. Herein, "2  monolayers.In this work, we have investigated the geometric, mechanical, electronic transport and optical properties of penta-PdQ2  monolayers were performed with in the SIESTA code35. The exchange–correlation functional approach was used in term of Perdew–Burke-Ernzerhof (PBE) type pseudopotential36. The basic unit cell of 2D-PdQ2  contains two Pd atoms and four Q  atoms as shown in Fig. Ry for PdS2 and PdSe2 monolayers were adopted, respectively. The doubled zeta plus (DZP) basis set was used with an energy of 0.02 Ry to expand the Kohn–Sham orbital. The Г- centred mesh of 20 × 20 × 1 and 15 × 15 × 1 k-points were sampled for PdS2 and PdSe2 monolayers, respectively under the Monkhorst–pack scheme in the 2D Brillouin zone37. The force 0.01 eV/Å was kept to relaxing the unstrained and strained structures of penta-PdQ2  monolayers. The phonon dispersive curves were calculated using the density functional perturbation theory (DFPT) formalism38. In all calculation, the Fermi level (EF) is shifted at the zero energy. The parameters of carrier mobility of penta-PdQ2  monolayers were found such as i.e., effective mass (m*), stiffness constants (C2D) and deformation energy (E1) approximations using the following formula proposed by the Bardeen and Shockley39, µ2D = e is elementary charge of an electron, ђ is the reduced Planck’s constant, C2D is the in-plane stiffness constant and for 2D system it is defined as S0 is cell area and a0 is the lattice constant at equilibrium, E and a are the total energy and lattice constant of the monolayer after deformation, T is the temperature (300 K). Also, m* is the effective mass in the transport direction and E1 is the deformation potential (DP) constant denoting the shifting of each band edges due to the applied biaxial strain εxy defined as Eedge is energy value of VBM (for holes) and CBM (for electrons).The structural, electronic transport and optical properties of 2D wrinkled PdQ2 monolayers are calculated by utilizing the non-equilibrium Green’s function (NEGF) in the form of I-V response by TRANSIESTA module of SIESTA code41. The electric current through the scattering region is calculated by exploring the Landauer-Buttiker formalism,bias) is the transmission probability of an electron incident at an energy E through the device under the bias voltage Vbias, 0 is the unit of quantum conductance. The optical properties were investigated including the dielectric function and absorbance coefficient by DFT within the random phase approximation (RPA)42. The dielectric function is given by ɛ (ω) = ɛ’ (ω) + i ε’’ (ω). Here, ε’(ω) and ε’’(ω) are the real and imaginary parts of the complex dielectric function43, respectively.The electronic transport properties of PdQ2  were systemitically relaxed and lattice structures of penta- PdQ2 were obtained. It is built up with two Pd and four Q  atoms in the unitcell as shown in Fig. Å), distance d (Å) between Pd-Q1, Pd-Q2 and Q1-Q2, thickness t (Å) and cohesive energy Ecoh (eV/atom) of PdQ2 monolayers.First, the geometric structures of pristine 2D penta-PdQa (b) of PdS2 and PdSe2 monolayers are 5.62 (5.42) Å and 5.94 (5.68) Å, respectively. The atomic distance between Pd-S1(Se1), Pd-S2(Se2), and S1(Se1)-S2(Se2) atoms are 2.35 (2.45) Å, 2.34 (2.42) Å and 2.14 (2.42) Å, respectively and shows good agreement with previously reported results34. To evaluate stability of resulted structures, the cohesive energy is calculated using the formula: Ecoh = Σ nX EX—EPdQ2/N. where EPdQ2 denotes total energy of PdQ2  unit cell, N is total atom in unit cell and nX and EX are number of atoms of each element and atomic energies of each atom in the unit cell, respectively. It is found that cohesive energies for PdS2 and PdSe2 monolayer are −4.70 and −4.30 eV/atom, respectively. Although this is larger than that for the 2D-antimony (−4.03), 2D-arsenene (−2.96), 2D-MoS2 (−4.11), 2D-MoSe2 (−3.94) and 2D-black phosphorous (−3.48). This is evidence that penta-PdQ2 monolayers have strong bonding in its ring-network. In addition, to confirm the kinetic stability of penta-PdQ2  structures, we calculated the phonon dispersion curves along high symmetric points for both PdS2 and PdSe2 monolayers and results are as shown in Figure S1 , ESI†. The highest phonon frequencies extended is up to 2275 cm-1 and 2193 cm-1 for PdS2 and PdSe2 monolayers, respectively. Evidently, no negative acoustics branch is appeared in the first Brillouin zone, itself proves the dynamical stability of both the monolayers at room temperature.As shown, in Table 2 and evaluated Young’s modulus Y (N/m) and bulk modulus B (N/m). The Young’s moduli Y (N/m) and bulk moduli B (N/m) were investigated under uniaxial and biaxial strain on the penta-PdQ2 monolayers. The range of applied strain x and y) on the penta-PdQ2 monolayer with following relation45,S0 is unstrained cross-sectional area of the unit cell. Es and A are the total strain energy and area of respected applied strain, respectively. Figure S2 , ESI† shows the strain energy vs area curve for pristine and loaded structures of PdS2 and PdSe2 monolayers, respectively. This curve is fitted by the polynomial curve fitting and with help of Eq. (B (N/m) as shown in Figure S2a,b, ESI†. The calculated bulk modulus (N/m) of PdS2 and PdSe2 are 30.22 and 23.56 N/m, respectively. These values are larger than that for the monolayer of Sb (~ 21.88 N/m) and As (~ 25.78 N/m)46, that indicate that penta-PdQ2 has stiffer and better resistance to deformation compared to antimony (Sb) and arsenene (As) monolayers. Afterwards, we have calculated the in-plane Young’s modulus Y (N/m) in longitudinal  or transverse  direction for penta-PdQ2. To obtained Young’s modulus from first-principles calculation, the following formula was employed45,Next, we focused on mechanical properties of penta-PdQmentclass2pt{minimS0 is unstrained cross-sectional area of the unicell. Next, 2 in the longitudinal or transverse direction. The Young’s moduli obtained by using the curves shown in Figure S3a,b, ESI†. The calculated Young’s modulus Y (N/m) along x (y) direction are 74.13 (42.40) N/m and 65.26 (28.62) N/m for penta-PdS2 and penta-PdSe2, respectively. The results indicating that, Young’s modulus decreases as we switched S to Se, and this is due to the increment in the Pd-S and Pd–Se bond strength. Xiong et al.47 had calculated orientation-dependent Young’s modulus of 2D orthorhombic MX2  and reported values are ~ 50 (37) N/m for PdS2 (PdSe2). From the present investigation of Young’s modulus of penta-PdQ2, we suggest that the 2D penta-PdQ2 can be preferred candidates for flexible devices because of having ultra-low values of Young’s modulus, compared to that of 2D graphene (340 N/m) and 2D MoS2 (125 N/m)49.Here, 2 monolayers and examined that up to which values of strain  that a monolayer can withstand prior to the fractured structure set. This can be calculated by the components of stress tensor with respect applied strain. The computed UTSH for penta-PdQ2 monolayers are shown in Fig. 2, the calculated values of UTSR along x (y) directions is 16GPa and 3.43 GPa, respectively  GPa. The present values of UTSR for penta-PdQ2 are higher than the blue-phosphorene monolayer  have also investigated. At PBE level, from Fig. 2 (PdSe2) is an indirect band gap with valence band maximum (VBM) and conduction band minimum (CBM) tracing at S- point (M-point) and Γ-point, respectively. The obtained indirect band gap of penta-PdS2 is EgPBE = 0.94 eV, while band gap for penta-PdSe2 is EgPBE = 1.26 eV as seen in Fig. 31. As DFT-PBE functional may underestimate the bandgaps of semiconductors, we also used GGA + U, LDA, LDA + U and HSE0652 approximations to ensure calculation reliability and the obtained indirect electronic bandgap of PdS2 (PdSe2) are 1.05 (1.26) eV, 0.92 (1.31) eV, 0.94 (1.30) and 0.92 eV, respectively. For the present computations, the Hubbard parameter value taken as 3.69 eV53. Next, having heavier element (Pd) in 2D penta-PdQ2 material, the spin–orbit coupling (SOC) effect may play a crucial role and could influence in the band gap (Eg) of material. Thus, we have also checked the SOC effect on the penta-PdQ2 monolayer and results are shown in Figure S4a,b, ESI†. Using PBE + SOC functional calculation, the obtained indirect band gap of penta-PdS2 is EgPBE+SOC = 1.16 eV, whereas band gap of penta-PdSe2 is EgPBE+SOC = 1.51 eV. Recently, the Yang et al.32, have reported electronic properties of penta-PdS2 from PBE (PBE + SOC) calculation and reported indirect band gap of 1.08 eV (1.13 eV), which is comparable with our results within of 12.9634, have reported a band gap of penta-PdSe2 with PBE level theory and mentioned indirect band gap of 1.38 eV, which is higher by 8.633, has computed indirect band gap of penta-PdSe2 monolayer via TB-mBJ-GGA potential and reported band gap 1.38 eV. The deviation of present band gap of penta-PdSe2 with PBE + SOC calculation is 92 monolayer to reach up the optimal value of 1.3 eV for solar cell applications, which is discussed in SQ Efficiency section. As presented in Fig. States/eV) are mostly ascend due to ‘d’ orbitals of Pd atom and ‘p’ orbitals of Q  atom in the penta-PdQ2 monolayer, which are also consistent to the past reported results34. Additionally, the TDOS difference from PBE to PBE + SOC calculations are shown in Figure S5a,b, (ESI†) for PdS2 and PdSe2 monolayers. we have also investigated PDOS (States/eV) as displayed in Fig. 2 and we conclude that the conduction bands (CBs) and valence bands (VBs) are highly occupied by the 4d-Pd states and 3p-Q  states, respectively.The band structure, total density of states (TDOS) and partial density of states (PDOS) of penta-PdQµ) of monolayer, which is one of essential factor for a high-performance device like FET. For this, we have exerted compressive  monolayers and traced influence on the band edges of CBM and VBM in the band structure, where system is anisotropic . The strength of strain along biaxial direction is given by54, S−S0)/S0]S0 is the unstrained lattice constant and S is the strained lattice constant of the monolayer. The response of applied strain to band gaps of penta-PdQ2 is shown in Fig. We further focused on the band alignment in terms of strain effect to drive the electronic properties. Simultaneously, we have also calculated carrier mobility  is transform into semi-metallic (~ 0 eV) by applying the biaxial compressive and tensile strain of −132 behaviour arises due to crossing one band of VBM at the Fermi level , ESI†). In contrary, up on the critical tensile (+ †). Additionally, we have also examined the phonon spectrum for each applied strain on penta-PdQ2 and computed its phonon frequency as summarized in Table S1, ESI†. Thus, our outcomes accomplish those electronic properties of studied 2D penta-PdQ2  materials are tuneable by applying biaxial strain, which establish that the penta-PdQ2 is a promising candidate for designing flexible nanodevices.As stated before, the fundamental indirect band gap of penta-PdS2 monolayers, we inspired to calculate the carrier mobility of penta-PdQ2 monolayers. For that the Bardeen and Shockley formalism39 had been applied, we have calculated the effective mass (m*), deformation potential E1 (eV) and stiffness constant C2D (J m-2) to check the flow of charge carrier along biaxial strain. The carrier mobility depends on the most influenced factor on the carrier effective mass m* of electron (e) and hole (h), that is directly derived from the electronic band structure as55,Owing to tunability of penta-PdQE1 is another vital parameter to effect carrier mobility and is obtained from the deformation theory (DP)39, which has been also successfully used method in previous reports for 2D MoS2, WS2 and phosphorene. The needed ingredients for carrier mobility (µ) are summarized in Table 2 (PdSe2) are 0.39 me (0.58 me) and 0.72 me (0.14 me), respectively. Our computed deformation potential E1 (eV) for penta-PdS2 is −6.44 eV for e and 6.84 eV for h, that is consistent with ones reported by Wang et al.17, along x or y direction. While in case of penta-PdSe2, the value of E1 is −2.91 eV for e and 6.14 eV for h that is also in accordance with the results of Qin et al.,33 for x or y direction. Whereas the in-plane stiffness constant C2D for penta-PdQ2 has anisotropic behaviour. The in-plane stiffness constant obtained for penta-PdS2 and penta-PdSe2 are 121.76 J m-2 and 94.62 J m-2, respectively. The stiffness constant C2D of penta-PdS2 is larger than the penta-PdSe2, indicating that PdSe2 monolayer is softer than the penta-PdS2 monolayer. As shown in Table 2 monolayer is 258 cm2 V−1 s−1, which is quite higher than the hole mobility ~ 67 cm2 V−1 s−1. Also, the carrier mobility ratio R comes out to be 0.26. Moreover, Wang et al.17 has reported the electron mobilities (cm2 V−1 s−1) of penta-PdS2 as 40.97 (x) and 169.11 (y), while hole mobilities (cm2 V−1 s−1) as 339.25 (x) and 91.73 (y). On other hand, if we compare carrier transport along biaxial strain of penta-PdSe2 the hole mobility is 1518 cm2 V−1 s−1 that is greater than the electron mobility ~ 442.49 cm2 V−1 s−1 and R is 3.43. However, it is observed that the hole mobility of penta-PdSe2 is higher than the 2D phosphorene56  and BN nanosheet 57, indicating that penta-PdSe2 would be a promising material for modelling electronic applications.Next, deformation potential 2 monolayers, we have examined the I-V characteristics, based on the equivalent transport theory41. Figure To understand the current sensitivity of the penta-PdQ2 and penta-PdSe2 monolayers are shown in Fig. 2 and PdSe2 monolayers, respectively. Apparently, it can be seen form Fig. 2 is more current-sensitive than the penta-PdSe2 monolayer. Although, in the case of penta-PdS2, the first peak in current is seen at 2.5 V with 14 µA and second current peak is located at 4.0 V with current of 13.2 µA. This is indicating a pronounced negative differential conductance effect (NDC)58 in the bias range 2.0–3.5 V  converters59.Here, we have taken 10 primitive cells, among them the lead had built up with four-unit cell (24 atoms) and central region consist of 12-unit cells (72 atoms). The I-V characteristic of both penta-PdSsee Fig. a. While see Fig. b. More p2  using Shokley-Queisser (SQ) limit that gives the percentage of power converted to electrical energy61. For this we required several parameters such as short circuit current density  of penta-PdQEg) plays a pivotal role in the calculation of the material efficiency. Moreover, the maximum possible SQ efficiency limit is specified of 33.7066. As seen from the Table 2 and penta-PdSe2 is observed and are 30.79 and 33.542 is larger than the efficiency of benchmark GaAs (~ 31.42 the efficiency at certain strain is higher as compared to the pristine (0 2) and −33.94 2). On other hand, under tensile strain the maximum efficiency arises that of 25.72 2 and 33.63 2, which is lower than with respective each of pristine  and perpendicular (Ec) electric vector (E). The computation is carried out using the Kramers-Kroning (KK) relationship67 and results are shown in Figs. In order to evaluate light absorption ability of penta-PdQħω) from 0 to 10 eV and absorption coefficients VIS; 1.59 – 3.26 eV) and near UV  spectrum regions are shaded by the light-pink and light-green colour, respectively. Figure 2, that simply gives the idea about the electronic polarizability of the material from the Clausis—Mossotti 68 relation. It can be seen from the Fig. 2 is , while static dielectric constant of penta-PdSe2 is 4.0  region for both 2, it is clearly seen  at ħω = 2.04 eV and 2.76 eV due to the electronic transition of ‘4p’ states of Se atom and ‘4d’ states of Pd atom between conduction bands (CBs) and valence bands (VBs) of the electronic band structure. Apparently, the peaks tendency of 2 monolayers.In present work, the computed dielectric constants are shown in the large range of photon energy (2 were recorded by absorption coefficient eV) as shown in Fig. 2, the first absorption peak orientation is in the ~ 1.5–5 eV and second broad peak’s orientation is cover the UV region up to photon energy of 16 eV. For penta-PdS2, the low energy excitonic peak occur around 2.1 eV in infrared region (IR) and that peak rise towards the visible region at 3.78 eV for both the polarization. The maximum absorption coefficient 2 for parallel and perpendicular polarization is 3.65 × 105 and 4.04 × 105, respectively , ESI† and S9 , ESI†. In penta-PdS2, we observed static refractive index as (†). With the parallel polarization, the ħω = 1.78 eV, then going down up to energy of 2.32 eV. Similarly, in case of perpendicular polarization it has also gone down in the VIS region with 1.23 of n (ω) by the same spectrum photon energy. Moreover, the reflectivity †. The static reflectance , ESI†. While, for penta-PdSe2, the static refractive index  monolayers. The positive phonon spectrum gives the ground state dynamical stability of both the materials. The obtained electronic band gap is 0.94 eV and 1.26 eV for penta-PdS2 and penta-PdSe2, respectively. From mechanical stability point of view, penta-PdS2 withstands up to 16x (y) directions, while penta-PdSe2 is up to 17I-V curve shows the NDC effect beyond the bias voltage of 2.5 V (for PdS2) and 3.0 V (for PdSe2), hence this feature leads us to conclude that penta-PdQ2 monolayers will be consider in future as promising material for NDC-based nanodevices. More importantly, the SQ efficiencies for pristine PdS2 and PdSe2 were 30.792 is very broad in UV regions. The maximum absorption coefficient 2 and penta-PdSe2 are found in the UV region are 4.04 5 and 5.28 5, respectively. Collectively, due to unique electronic, mechanical, transport and optical properties of penta-PdQ2 monolayers, the 2D materials have application prospect in the arena of semiconducting nanodevices.In the summary of the work, the first principles method has been employed to study the band structure, bulk modulus, Young’s modulus, transport properties such as carrier mobility and Supplementary Information.


Dictyostelium has proven to be a valuable system for dissecting the molecular mechanisms of cell polarity. Previous studies in Dictyostelium have revealed a range of signaling and cytoskeletal proteins that function at the leading edge to promote pseudopod extension and migration. In contrast, how proteins are localized to the trailing edge is not well understood. By screening for asymmetrically localized proteins, we identified a novel trailing-edge protein we named Teep1. We show that a charged surface formed by two pleckstrin homology (PH) domains in Teep1 is necessary and sufficient for targeting it to the rear of cells. Combining biochemical and imaging analyses, we demonstrate that Teep1 interacts preferentially with PIP2 and PIP2in vitro and simultaneous elimination of these lipid species in cells blocks the membrane association of Teep1. Furthermore, a leading-edge localized myotubularin phosphatase likely mediates the removal of PIP2 from the front, as well as the formation of a back-to-front gradient of PIP2. Together our data indicate that PIP2 and PIP2 on the plasma membrane jointly participate in shaping the back state of Dictyostelium cells.Polarity, which refers to the molecular or structural asymmetry in cells, is essential for diverse cellular functions.  Dictyostelium discoideum has provided important insights into the mechanisms underlying the establishment and maintenance of cell polarity  or trailing edge (back) of migrating cells, creating functionally distinct ends that promote cell migration. Events that occur at the leading edge include the activation of several Ras and Rac family GTPases, activation of mTORC2 and its substrates of the Akt/PKB family kinases, accumulation of the class I PI3-kinases (PI3Ks) and their product PIP3, and recruitment of a number of regulators of actin polymerization, such as the Scar/WAVE and Arp2/3 complexes responsible for pseudopod projection  domain-containing proteins, which regulate leading edge activities P2, exhibit complementary patterns of localization P2 are fairly modest in migrating cells P2 has been suggested to regulate back events, largely based on study of the trailing-edge protein CynA and its PH domain-containing region P2 and PIP2 act independently or cooperatively.Compared to the well-characterized front state, the molecular definition of the back state remains obscure. PIPe furrow . In linelization . Furtherck state . Howeverng cells , which sg region . Evidencegion P2 to modulate the posterior accumulation of Teep1 and to shape the back state of cells.Compared to the number of leading-edge proteins identified thus far, few proteins have been found at the trailing edge, and even fewer have been found to exhibit behavior similar to back proteins in the absence of an intact actin cytoskeleton . This prlgorithm . Over 50lgorithm . This ap3/PIP2 reporter PHcrac , and CynA , the locN411) with an equivalent level of expression, extent of rear enrichment, and responsiveness to stimulation as the full-length protein P2, which has been implicated in back protein localization P2 and reduce the membrane-to-cytosol ratio of CynA P2 sensor TAPP1 in Dd5P4− cells Plization , underpi of CynA . We geneut cells . The newl strain , and thel strain . As seenl strain , we obse− cells . In addi− cells . However− cells , indicat2. The accumulation of PIP2 at the back of migrating cells and the cleavage furrow have been observed in Dictyostelium and other systems  and Nodulin (the Nlj6-like nodulin domain of AtSfh1) P2 to guide the dissociation of Teep1 from protrusions and following stimulation.Next, we investigated PIP systems . Consisttrusions . However AtSfh1) , we noti AtSfh1) . A fracttic cups  or pseudtic cups , but Teelocation . Therefo2 and PIP2 (2 and PIP3 as expected (N380) containing the PH domains as a GST-fusion protein P2, and PI5P P2, and a modest increase was observed with those containing PI5P P2 in the liposomes further increased the association P2 P2 PIP2 . PHcrac-expected  P2 . MoreoveIP2 , suggest2 is consistent with the existence of a back-to-front gradient of PIP2 on the plasma membrane, but binding to PIP2 is rather unexpected. Although PIP2 on the plasma membrane has been linked to activities such as mTORC1 activation P2 are required to target Teep1.The ability of Teep1 and Pten to bind PIPtivation , this lilular PI . The lipDictyostelium, as in various other systems, most cellular pools of PIP2 depend on the activity of the phosphoinositide 5-kinase PIKfyve, which produces PIP2 by phosphorylating PI3P P2 is produced mainly by the PIP5 kinase PikI P2 simultaneously, we used a chemically inducible dimerization system P2 on the plasma membrane within 10–15 min P2 blocks the membrane association of Teep1.To reduce the levels of PIPn system . In the 0–15 min . We tranWT cells , with thWT cells . In contof Teep1 , with thof Teep1 . The mag similar , validat2-sensor , we further analyzed the involvement of PIP2 in back protein localization. The PIP2-binding phox homology (PX) domain localized to endosomal structures when expressed as a tandem dimer in cells P2 regulate the posterior accumulation of proteins, such as Teep1, by jointly shaping the back state of the plasma membrane.Using a newly isolated PIPin cells . Interesin cells . Collect2 that acts together with PIP2 gradient to regulate back events. The gradient of PIP2 on the plasma membrane is thought to be established by PI5K- and Pten-mediated production at the back, as well as PI3K- and PLC-mediated removal from the front P2 turnover. This process is proposed to be catalyzed by the Sac1-related phosphatase Fig4 or myotubularin family of phosphatases  and nine putative myotubularins, which we named Mtm1-9 Phe front . We specfyve-GFP  was locafyve-GFP , suggestphatases . Dictyosd Mtm1-9 . We tagg3. We found that the plasma membrane localization of Mtm6 was abolished in a quintuple PI3K mutant P2, with a preference for PIP3 P2 during macropinosome formation P2. In line with this model, a malachite-based assay revealed that Mtm6-GFP immunoprecipitated from cell lysates exhibited high activity against PIP2 PIP2 . Mtm6-GFIP2 , and theut cells . Opposit. Consistently, when plated on non-nutrient agar, teep1− cells differentiated and formed streams of migrating cells and fruiting bodies similar to WT P2 regulate the posterior accumulation of proteins, such as Teep1, by jointly shaping the back state of the plasma membrane. First, we found that a potential charged surface formed by the two N-terminal PH domains is both necessary and sufficient for targeting Teep1 to the rear of cells P2 and PIP2 P2 partially impairs the plasma membrane association of Teep1, whereas simultaneous elimination of PIP2 and PIP2 nearly blocks its membrane localization P2-recognition modules exhibits a posterior enrichment P2 from the front and the formation of a reverse PIP2 gradient Pof cells . Second,IP2 . Third, lization . In addirichment . Finallygradient . Consistof Teep1 . Mtm6 isTeep1 P2, suggesting that its localization may be regulated by a coincidence-detection mechanism, but we do not yet understand how this is achieved at the molecular level. We found that PIP2 or PIP2 depletion has different impacts on the localization of Teep1 bearing mutations in either of the PH domains P2 simultaneously. Assessing the lipid binding specificity of individual PH domain in cells or in vitro assays is needed to distinguish between these possibilities. However, the expression of the two PH domains is poor when expressed separately in cells or as recombinant proteins, precluding further analysis.Our study raises a few intriguing questions that remain to be answered. First, Teep1 seems to interact with both PIP domains . This ob domains . The PH  domains . Thus, c2 and PIP2 provide a general mechanism for targeting proteins to the trailing edge requires further investigation. Using purified proteins, we found that Pten also binds preferentially to liposomes containing PIP2 and PIP2 P2 by recruitment of Inp54 in pikfyve− cells not only results in Teep1 dissociation but also causes substantially increased blebbing P2 may have a greater impact on the back state of cells by affecting the membrane association of multiple proteins and reducing the threshold for blebbing.Second, whether reverse gradients of PIPIP2 , suggestblebbing , a phenoblebbing . In contblebbing . Considemulation , the rem2/PIP2 is unlikely to be the only back signal. For example, the back-localized CynA protein has been shown to interact selectively with PIP2 PIP2 . The locIP2 . It willpikI− cells were cultured on bacterial lawns (Klebsiella aerogenes) and transferred into HL5 before use in experiments (WT cells were derived from the Ax2 (Ka) axenic strain . All generiments . Cells ceriments .teep1, mtm6, pikfyve, and Dd5P4 deletion, a blasticidin S resistance (BSR) cassette was inserted into pBlueScript II SK+ to generate pBlueScript-BSR  was added between the two PX domains. To express the chimeric sensor, the Nodulin domain of Atsfh1 was amplified using primers F3 and R3 and inserted into the PX dimer-GFP construct at the SpeI site.To generate constructs expressing GFP- or RFP-fusion proteins, DNA fragments were PCR-amplified using primers listed in 5 cells were plated in 8-well coverslip chambers  filled with HL5 or LoFlo medium (Formedium) and allowed to adhere. Images were acquired on a Zeiss 880 inverted microscope equipped with a 40 ×/.95 or 63 ×/1.4 oil-immersion objective. For LatA treatment, cells were incubated with 5 μM LatA (Enzo Life Sciences BML-T119-0100) for 5–10 min before imaging. To image the localization of Teep1 in LatA-treated cells in response to cAMP gradient, a μ-Slide Chemotaxis chamber (ibidi) was utilized. Cell loading was conducted following the manufacturer’s instruction. Before imaging, 1 μl of LatA (50 μM) was added via port A. To image protein translocation in response to cAMP stimulation, differentiated cells were stimulated with 1 μM cAMP. To image protein translocation in response to folic acid, vegetative cells were incubated in development buffer (DB) for 30 min before the addition of 200–500 μM folic acid. The Inp54 recruitment experiment was performed as described previously  and analyzed using Ibidi chemotaxis tool software.For random motility assay, vegetative cells were seeded in culture plate in HL5 and allowed to adhere for 4 h. Before imaging, the medium was replaced with fresh HL5. Images were acquired at 20 s intervals with phase illumination using a 10×/.45 or 20×/.8 objective. Under-agarose folic acid chemotaxis assay was performed as described before . BrieflyEscherichia coli BL21 cells transformed with GST-Teep1N380, GST- Teep1N380M, or GST-Pten were grown until absorbance at 600 nm of 0.8 and induced with 0.4 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) for 16–18 h at 20°C. Bacteria pellet was resuspended in ice-cold buffer A  and lysed with a high-pressure homogenizer. Cell suspension was centrifuged at 15,000 g for 30 min to pellet the debris. The supernatant was incubated with glutathione sepharose beads  for 2 h at 4°C. The protein of interest was eluted with elution buffer  followed by gel filtration on a 10/300 G200 Superdex column  equilibrated with buffer B . The GST tag was removed by PreScission protease digestion on the column. The cleaved protein was collected and applied to gel filtration column for further purification. Fractions from the gel filtration column were pooled and concentrated.Lipid strips  were obtained from Echelon. Dot blot assay using cell lysates was performed as described before . When th2 (850153), PIP2 (850154), PIP2 (850155), and PIP3 (850156) were obtained from Avanti Polar Lipids and dissolved in chloroform. POPC, NBD-PE, and variable PIPs were mixed at molar ratio of 97:1:2 (for binding with Teep1) or 89:1:10 (for binding with Pten). Mixed lipids were dried under a flow of nitrogen gas and in SpeedVac for 1–2 h. The lipid films were resuspended in Hepes-NaCl buffer (50 mM Hepes pH7.0 and 150 mM NaCl) to a final concentration of 5 mM and subjected to freeze-thaw cycles 11 times. Unilamellar liposomes were generated via extrusion through a nanopore membrane with a pore size of 100 nm (Avanti Polar Lipids 610005); the process was repeated 11 times. The liposomes were mixed with purified proteins at molar ratio of 1,000:1 in a 50 μl reaction and incubated at 4°C for 1 h with gentle agitation. 30 μl of the protein-liposome mixture was diluted with 100 μl 1.9 M sucrose, placed at the bottom of a centrifugation tube, and overlaid sequentially with 100 μl 1.25 M sucrose and 20 μl Hepes-NaCl buffer. The sucrose gradient samples were centrifuged at 174,000 g for 1 h at 4°C. Five fractions were collected from the top, mixed with SDS loading buffer, and subjected to SDS-PAGE followed by silver staining. Relative binding was calculated as the sum of band intensities of top two fractions divided by the sum of band intensities of all five fractions.POPC (850457), NBD-PE (810145), PI3P (850150), PI4P (850157), PI5P (850152), PIP2, and 5% Glycerol). Beads containing 200 ng Mtm6-GFP were incubated with 3,000 pmol substrate in a 25 µl reaction for 30 min at 22°C. 20 μl supernatant was mixed with 80 μl Malachite Green solution at room temperature for 30 min. Free phosphate released was measured at 620 nm wavelength.The phosphatase activity of Mtm6 was determined using a Malachite green assay kit . Cells expressing Mtm6-GFP were starved without cAMP pulses for 3 h. Cells were washed with wash buffer (20 mM Hepes pH7.2 and 150 mM NaCl), resuspended in lysis buffer , and incubated on ice for 5 min. Lysates were centrifuged for 10 min at 4°C. The supernatant was incubated GFP trap beads (Smart-Lifesciences) for 1 h at 4°C. Beads were washed with wash buffer and reaction buffer  and incubated for 10 min on ice. Lysates were centrifuged for 10 min at 4°C. The supernatants were incubated with GFP-Trap beads for 1 h at 4°C. Beads were washed with lysis buffer. Sample were eluted with SDS loading buffer and subjected to SDS-PAGE followed by mass spectrometry analysis or immunoblotting. Mass spectrometry analysis and immunoblotting were performed as described before (For immunoprecipitation assays, cells were starved without cAMP pulses for 3 h. Starved cells were lysed in lysis buffer (10 mM NaPi pH 7.2, 100 mM NaCl, 0.5% NP-40, 10% Glycerol, 1 mM NaF, .5 mM Nad before . Anti-GF5 cells were plated in 6-well tissue-culture plate for 8 h. 2 ml fresh medium was added before the plate was placed on an orbital shaker and rotated at 200 rpm for 1 h. Floating and adherent cells were then counted to calculate the percent of adherent cells.6×10p < .001, **p < .01, *p < .05, ns not significant.Statistical analysis was performed using GraphPad Prism. Statistical significance was determined by unpaired t test or one-way ANOVA with Dunnett or Tukey post-test. In all figures, *** indicates