There are six known families of homing endonucleases, LAGLIDADG, GIY-YIG, HNH, His-Cys box, PD-(D/E)-XK, and EDxHD, which are characterized by their conserved residues. Previously, we discovered a novel homing endonuclease F-CphI encoded by ORF177 of cyanophage S-PM2. F-CphI does not resemble any characterized homing endonucleases. Instead, the C-terminus of F-CphI aligns well with the N-terminal catalytic domain of a Holliday junction DNA resolvase, phage T4 endonuclease VII (Endo VII).A PSI-BLAST search resulted in a total of 313 Endo VII motif–containing sequences in sequenced genomes. Multiple sequence alignment showed that the catalytically important residues of T4 Endo VII were all well conserved in these proteins. Our site-directed mutagenesis studies further confirmed that the catalytically important residues of T4 Endo VII were also essential for F-CphI activity, and thus F-CphI might use a similar protein fold as Endo VII for DNA cleavage. A phylogenetic tree of the Endo VII motif–containing sequences showed that putative resolvases grouped into one clade while putative homing endonucleases and restriction endonucleases grouped into another clade.Based on the unique conserved residues, we proposed that F-CphI represents a new homing endonuclease family, which was named the DHHRN family. Our phylogenetic analysis could be used to predict the functions of many previously unknown proteins.The online version of this article (10.1186/s13100-018-0132-5) contains supplementary material, which is available to authorized users. Many group I introns contain open reading frames (ORFs) that encode homing endonucleases. In a process called intron-homing, a homing endonuclease cleaves an intronless allele near the intron insertion site (IIS) and repair of the DNA break using the intron-containing allele as template transfers the intron and the homing endonuclease gene into the intronless allele . Homing Homing endonucleases have been grouped into six families, LAGLIDADG, GIY-YIG, HNH, His-Cys box, PD-(D/E)-XK, and EDxHD, which were named for the conserved amino acid residues (reviewed by ). CrystapsbA gene ATP and T4 polynucleotide kinase) before being used in annealing reactions with an unlabeled partner. Labeled duplexes were purified using a QIAquick PCR Purification Kit (Qiagen). 10 nM labeled duplex was incubated with 1 μM F-CphI in the optimal endonuclease assay buffer at 25 °C. Reaction products were extracted with an equal volume of phenol and separated on a 4% denaturing polyacrylamide gel. Sequences of SBM4-60Top and SBM4-60Top-r are listed in Additional file Oligonucleotide duplex containing the F-CphI cleavage site  was geneTo compare the DNA binding ability of F-CphI wild type and mutants, both specific and nonspecific DNA duplexes were used. A specific oligonucleotide duplex containing the F-CphI cleavage site was generated by annealing 5′ end labeled SBM4-60Top and unlabeled SBM4-60Top-r. Nonspecific competitor duplex was generated using oligonucleotide endoV41A and its complementary sequence endoV41comp . Band intensity of DNA-bound complex at protein concentration of 0 nM was used as the background signal, and was subtracted from the signal intensities obtained from all the bands. The fraction of DNA bound was thus calculated from bound/(bound + unbound), and was plotted versus the concentrations of F-CphI wild type, and D101N and H102T mutants, respectively. The data were then fitted by non-linear regression model with the nlsLM function of minpack. lm package in R, using the following equation modified from previous studies [Quantitation of band intensities from the electrophoretic mobility shift assay were performed in the GelQuant.NET software . In each search, we performed BLASTp and PSI-BLAST with two iterations and set a final e-value cut-off of 1E-4. In search of the Endo VII homologs, both the catalytic domain of T4 Endo VII (residues 1–97) and the corresponding sequence of F-CphI (residues 62–139) were used as the query protein sequences. The resulting 313 protein sequences from complete genomes were fetched from NCBI for further analysis. In search of the HNH and His-Cys box family proteins, the catalytic domains of I-HmuI (residues 49–97) from Bacillus phage SP01 and I-PpoI (residues 93–127) from Physarum polycephalum were used as the query sequences, respectively. Sequences from incomplete genomes or obtained from environmental samples were discarded prior to protein sequence extraction.In-house BLAST searches were carried out against the downloaded non-redundant (nr) protein database (http://espript.ibcp.fr/) to obtain the consensus sequence with conserved amino acids highlighted.MAFFT  and Clushttp://skylign.org), which is based on the profile hidden Markov model (HMM) analysis [In order to profile the consensus sequence from the multiple sequence alignment, we generated sequence logo on the Skylign website .ProtTest  was usedAdditional file 1:Figure S1. Purification of F-CphI wild type and mutants. (A) E. coli cells were induced to express F-CphI and then disrupted by sonication. The crude lysate was centrifuged and the supernate was loaded on a HisTrap FF crude column. Five elutions (1 ml each) were collected. The crude lysate, supernate, pellet, and five elutions were separated on 15% SDS-PAGE. (B) Purified F-CphI wild type and mutants (second elutions) were separated on 15% SDS-PAGE. (PDF 233 kb)Additional file 2:Table S1. Endo VII motif-containing proteins. Proteins containing the Endo VII motif were revealed by PSI-BLAST searches. In the last column, the position of the Endo VII motif in a protein is shown. The Endo VII motif of a protein could be in the C terminus (C), the N terminus (N), or in the middle (indicated by residue numbers/full length of a protein). In the same column, F-CphI and Endo VII indicate that a protein aligns with the full length of F-CphI or Endo VII, respectively. (XLSX 29 kb)Additional file 3:Figure S2. Multiple sequence alignment of the Endo VII motif sequences. Dots represent gaps in the alignment. Totally conserved residues at a position are shown by white letters in red background. Residues with conservation above 70% at a position are shown by red letters in blue boxes. The position numbers of F-CphI and Endo VII residues are shown on top of the alignment. (PDF 1993 kb)Additional file 4:Figure S3. Gel shift assay of F-CphI wild type and mutants. (A) 2 nM 32P labeled 60 bp duplex containing the F-CphI recognition site was incubated on ice for 15 min with increasing concentration of each protein. The free DNA (F) and protein-bound complexes (C) were separated on 8% native polyacrylamide gel. (B) 2 nM 32P labeled 60 bp duplex containing the F-CphI recognition site and 200 nM protein were incubated with increasing concentration of unlabeled non-specific 60 bp duplex (0 nM to 20 nM). The first lane in each gel shows the pattern of free DNA (no protein was added in the reaction). (C) Using the gels in A, the fractions of protein-bound complexes for wild type, D101N, and H102T were plotted against protein concentrations. The binding curves were generated from the non-linear regression fitted data, and were used to estimate the apparent equilibrium dissociation constant (Kd). Kd values are shown in each graph and errors represent 95% confidence interval. (PDF 126 kb)Additional file 5:Figure S4. Sequence logos of the DHHRN, HNH, and His-Cys box endonuclease families. Sequences near the active site of an endonuclease family are used to generate sequence logos. At each position of the sequence logo, the total height of a stack of letters shows the information content in bits that is calculated from a profile hidden Markov model, and the height of a letter relative to the total height of letters at a position represents the letter’s frequency. The red lines indicate gaps in the multiple sequence alignment. Above each sequence logo, the corresponding residue numbers of Endo VII, I-HmuI, and I-PpoI are shown, which are representatives of the DHHRN, HNH, and His-Cys box families, respectively. Black boxes show the corresponding catalytic residues used by Endo VII, I-HmuI, and I-PpoI. (PDF 351 kb)Additional file 6:Table S3. Oligonucleotides used in this study . (DOCX 12 kb)Additional file 7:Table S2. Clades of Endo VII motif-containing proteins as shown in Fig.


Saccharomyces cerevisiae EG110 yeast cells, but not of the wild-type cells, grown aerobically in ethanol medium. This occurred at both the gene level, as indicated by microarray and qRT-PCR analyses, and at the protein level as indicated by enzyme assays. These results show that in redox-compromised MnSOD-deficient yeast cells, but not in wild-type cells, aspirin starves the mitochondria of acetyl-CoA and likely causes energy failure linked to mitochondrial damage, resulting in cell death. Since acetyl-CoA is one of the least-studied targets of aspirin in terms of the latter’s propensity to prevent cancer, this work may provide further mechanistic insight into aspirin’s chemopreventive behavior with respect to early stage cancer cells, which tend to have downregulated MnSOD and are also redox-compromised.Aspirin is a widely used anti-inflammatory and antithrombotic drug also known in recent years for its promising chemopreventive antineoplastic properties, thought to be mediated in part by its ability to induce apoptotic cell death. However, the full range of mechanisms underlying aspirin’s cancer-preventive properties is still elusive. In this study, we observed that aspirin impaired both the synthesis and transport of acetyl-coenzyme A (acetyl-CoA) into the mitochondria of manganese superoxide dismutase (MnSOD)-deficient  These are attributed not only to aspirin’s anti-inflammatory and anti-platelet effects, but also to its propensity to induce programmed cell death, such as apoptosis, in cancer cells5. The full range of mechanisms underlying aspirin-induced apoptosis of malignant cells is still not fully understood, and has aroused considerable research efforts involving the use of several eukaryotic experimental models. One such model is the yeast Saccharomyces cerevisiae, long used to study apoptosis in living organisms since it retains several core eukaryotic cellular processes, including the hallmark features of apoptosis9. In particular, yeast serves as a valuable tool to study mitochondria-associated regulated cell death12.Aspirin  is a widely used anti-inflammatory, cardioprotective and antithrombotic drug that has, in recent years, been found to have promising chemopreventive, antineoplastic properties13, we decided to use the redox-compromised, MnSOD-deficient S. cerevisiae EG110 cells as a model of cancer cells. In fact, cancer cells tend to manifest downregulated MnSOD during the early stages of their development16 and are also redox-compromised17.Since mitochondrial manganese superoxide dismutase (MnSOD) deficiency can reprogram cells to a cancer prone form of metabolism (Warburg effect)S. cerevisiae cells, but not in wild-type yeast cells, during aerobic growth in ethanol medium21. We also showed that the aspirin-induced apoptosis in redox-compromised MnSOD-deficient yeast cells is associated with severe mitochondrial dysfunction, including events such as overproduction of mitochondrial superoxide radicals (O2˙−), oxidation of mitochondrial NAD(P)H21, decreased respiration, release of cytochrome c and disruption of the mitochondrial membrane potential20. Overall, these results indicated that the mitochondrial milieu constitutes a critical cellular target of aspirin.In our previous studies, aspirin reproducibly and robustly induced an apoptotic phenotype in MnSOD-deficient 2. Hence, this work provides novel mechanistic insight into the metabolic changes associated with aspirin-mediated apoptosis. This may be of relevance to aspirin’s chemopreventive behavior in redox-compromised cancer cells during their early stages of development.In this study, we examined the effect of aspirin on the expression of genes involved in acetyl-coenzyme A (acetyl-CoA) generation and its transport into the mitochondria of MnSOD-deficient yeast cells growing aerobically on ethanol medium. Although acetyl-CoA is a metabolite of fundamental importance in eukaryotic organisms, since it drives the tricarboxylic acid (TCA) cycle required for energy generation, it remains one of the most poorly studied potential targets of aspirin in the context of the latter’s antineoplastic effectsSaccharomyces cerevisiae EG103 (MATα leu2-3 112 his3Δ1 trp1-289a ura3-52 GAL+) and the MnSOD-deficient yeast strain EG110 (EG103 sod2 Δ:: TRP1), kindly provided by Edith Gralla, University of California, Los Angeles and Valeria C. Culotta, Johns Hopkins University, Baltimore.The yeast strains used in this study include the wild-type ADH2-overexpressing strains (EG103 pGPD-ADH2 and EG110 pGPD-ADH2) the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (pGPD) was introduced to the endogenous ADH2 locus according to the method described by Janke et al.22. The plasmid pYM-N15 served as the template to generate a linear PCR fragment with primers 5′-ACAAAAAGCATACAATCAACTATCAACTATTAACTATATCGTAATACACAATGCGTACGCTGCAGGTCGAC-3′(ADH2_S1) and 5′-TTGCCGTTGGATTCGTAGAAGATAATGGCTTTTTGAGTTTCTGGAATAGACATCGATGAATTCTCTGTCG-3′(ADH2_S4 primer). Strains carrying an HA-tagged version of Adh2 were generated by similar approaches using pYM-N16 (EG103 pGPD-3HA-ADH2 and EG110 pGPD-3HA-ADH2) or pYM-N14 (EG103 ADH2-6HA and EG110 ADH2-6HA) as templates and primers ADH2_S1 and ADH2_S4 or 5′-GGCATACTTGATAATGAAAACTATAAATCGTAAAGACATAAGAGATCCGCTTAATCGATGAATTCGAGCTCG-3′ (ADH2_S2) and 5′-AGATGGAGAAGGGCCAAATTGCTGGTAGATACGTTGTTGACACTTCTAAACGTACGCTGCAGGTCGAC-3′(ADH2_S3), respectively. Correct integration of linear DNA fragments was verified by colony PCR using primers 5′-CCTGTGTAACTGATTAATCCTGC-3′ (ADH2_up) and 5′-GTCGACCTGCAGCGTACG-3′ (S1/S3_reverse) for all N-terminal integrations and primers 5′-GGAAGAATTGTTTACCTCGCTC-3′ (ADH2 -fw) and S1/S3_reverse for C-terminal tagging.To generate Yeast cells were aerobically cultivated in YPE medium (1% (w/v) yeast extract, 2% (w/v) bactopeptone and 3% (v/v) ethanol) in the absence and presence of 15 mM aspirin  (Sigma-Aldrich), at 28 °C with constant shaking at 250 rpm. The pH of the aspirin-treated YPE medium was adjusted to 5.5 using 1 M Trizma base (Sigma-Aldrich), prior to inoculation. For plates, cells were cultivated on YPD medium (1% (w/v) yeast extract, 2% (w/v) bactopeptone and 2% (w/v) glucose) containing 2% (w/v) agar, at a sustained incubation temperature of 28 °C.600). Cell viability was measured by plating serial dilutions of yeast cell cultures onto YPD plates (500 cells per plate). The plates were left to incubate for at least 48 h at 28 °C after which colony forming units (cfus) were counted. The cfu counts of aspirin-treated cells were then presented as fold changes in viability by normalizing them to the cfu counts of untreated cells.Cell growth was measured as the optical density at 600 nm (ODYRA1 gene using the custom-designed primers 5′-TGTCGGTGGTACTCGTGGTA-3′ (YRA1-F) and 5′-TAGTCCGCCATTTCCTTGTC-3′ (YRA1-R), at an annealing temperature of 54 °C, together with the Thermo Scientific 2X ReddyMix PCR Master Mix. The RT-PCR products were then visualized by electrophoresis on a 2% (v/v) agarose gel under UV light.Yeast cells were aerobically cultivated for 48 h (mid-log phase) in YPE medium in both the absence and presence of 15 mM ASA, at a temperature of 28 °C with constant shaking at 250 rpm. Harvested yeast cells were mechanically lysed by beadmilling, using a micro-minibeadbeater (Biospec), in the presence of acid-washed glass beads (500 nm diameter) and lysis buffer (RLT) provided by the Qiagen RNeasy Mini Kit. Three cycles of 60 s beadmilling followed by 60 s of cooling on ice were carried out. Total yeast RNA was extracted and purified using the Qiagen RNeasy Mini Kit, according to the manufacturer’s instructions. The quantity and quality of total RNA was determined using a Thermo Scientific Nanodrop spectrophotometer and an Agilent Bioanalyzer. Moreover, the RNA samples were subjected to reverse transcription polymerase chain reaction (RT-PCR) in order to verify that they were suitable for complementary DNA (cDNA) synthesis and not significantly contaminated by guanidine salts or ethanol, which interfere with the downstream microarray hybridization. One microgram of the RNA was used to synthesize cDNA using a Quantitect Reverse Transcription Kit (Qiagen). The cDNA was probed for the constitutive, intron-spanning 23, implemented with AltAnalyze Version 2.0.8.1 (University of Cincinnati).Preparation of complimentary RNA and subsequent hybridization to whole yeast genome microarrays were carried out using the GeneChip Yeast Genome 2.0 Array (Affymetrix). Normalization of the resulting microarray data was carried out using the robust multi-array average (RMA) methodt-statistics and corresponding P-values were determined for the probe sets of every individual S. cerevisiae gene present on the microarray chip. Differentially expressed genes with Benjamini-Hochberg-adjusted P-values < 0.05 and fold changes ≥1.5 (in presence of ASA versus absence of ASA) were identified. Microarray hybridization and analysis were carried out on three biological replicates.Normalized log2-expression values, fold changes, moderated 24.The normalized microarray data sets were used for principle components analysis (PCA) to identify correlations and outliers among individual aspirin-treated and untreated samples. The normalized datasets were further analyzed by hierarchical cluster analysis. Both PCA and hierarchical clustering were carried out using the online ClustVis web toolP-value < 0.05.Identification of enriched biological process, molecular function and cellular component GO categories was carried out by over-representation analysis (ORA) using GO-Elite software Version 1.2.6, which is integrated with AltAnalyze. Statistical significance of GO enrichment was determined using the Fisher Exact Test with a threshold All microarray datasets related to this study were deposited in the GEO (NCBI) repository and are accessible through the Accession number: GSE115660.S. cerevisiae target genes were determined by qRT-PCR to validate the microarray results. The extraction of total RNA from yeast cells, verification of its quality and the subsequent preparation of cDNA were carried out as described in a previous section. Each sample of resulting cDNA was probed in triplicate for target genes using the custom-designed oligonucleotide primer pairs (Integrated DNA Technologies) shown in Supplementary Table GLC7 and SMD2 reference genes as an internal control  of target genes as a measure of their differential expression in yeast cells grown in the absence and presence of aspirin, was carried out using qbase + (Biogazelle). Differentially expressed genes with significant P-values < 0.05 and fold changes >1.5 (in presence of ASA versus absence of ASA) were identified. The qRT-PCR assays were carried out on three biological replicates.The respective messenger RNA (mRNA) levels of differentially expressed 25, acetyl-coenzyme A synthetase (ACS) activity was measured according to van den Berg et al.26 and carnitine acetyl transferase (CAT) enzyme activity was quantified according to Chase27. Peroxisomal citrate synthase activity was measured according to Srere28 in yeast cytosolic fractions previously isolated from the mitochondria according to Glick and Pon29. The protein concentrations of total and cytosolic yeast cell extracts were determined with the Pierce Bicinchoninic Acid Assay Kit (Pierce), using bovine serum albumin (BSA) as a protein standard.Yeast total cell extracts, cultivated for 48 h in ethanol medium in the absence and presence of aspirin, were prepared by beadmilling, using a micro-minibeadbeater (Biospec), in the presence of acid-washed glass beads (500 nm diameter). Alcohol dehydrogenase (ADH) activity was measured according to Vallee and HocheImmunoblotting after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the cytosolic fractions to confirm the absence of any mitochondrial protein, was carried out using standard protocols, with antibodies specific for yeast cytosolic glucose-6-phosphate dehydrogenase  and mitochondrial heat shock protein 60 .30 was performed using standard protocols with antibodies specific for yeast glyceraldehyde-3-phosphate dehydrogenase  and HA .Immunoblotting after SDS-PAGE of chemically lysed yeastt-test . Multiple comparisons between treatments and controls were analyzed by one-way ANOVA with the post-hoc Bonferroni test. Where appropriate, the nonparametric Mann-Whitney U-test and Kruskal-Wallis test were applied to confirm the results of the t-tests and one-way ANOVA tests, respectively. Significance was accepted at a P-value < 0.05.Pairwise comparisons between treatments and controls were examined using the unpaired, two-tailed versus untreated EG110 sample groups was observed along principal component 2 (PC2), with a variance of 4.8%. This distinct clustering of aspirin-treated versus untreated EG110 sample groups was corroborated by the hierarchical clustering heatmaps  and MnSOD-proficient (EG103) yeast cells grown in ethanol medium. Principle Components Analysis (PCA) Fig.  of the naps Fig. , showingversus untreated biological replicates, along PC2  ontology  , but not in EG103 (MnSOD-proficient wild-type) yeast cells , but not in EG103, yeast cells 46. Both of these isoforms actively ligate acetate to CoA to yield acetyl-CoA, however, the expression of Acs1 is known to be heavily de-repressed in the absence of glucose and in cases where there is a prevalence of non-fermentable carbon sources, such as ethanol and acetate47. Moreover, the affinity of Acs1 for acetate is reported to be several times greater than that of the constitutively expressed Acs2 isoform, suggesting that Acs1 is primarily responsible for acetyl-CoA synthesis during growth on non-fermentable carbon sources such as ethanol26. This implies that the aspirin-induced decrease of acetyl-CoA synthetase activity observed in redox-compromised EG110 yeast cells, largely reflects an impairment of Acs1 activity. This does not necessarily exclude an accompanying aspirin-induced impairment of Acs2 activity. However, the aspirin-induced decline in ACS2 gene expression was less than 1.5-fold, as opposed to the 2.72-fold downregulation of ACS1.In agreement with the microarray and qRT-PCR results, aspirin significantly decreased the overall acetyl-CoA synthetase enzyme activity in EG110, but not in EG103, yeast cells grown for 48 h in ethanol medium Table . There a48. Hence, the reduced activity of CAT enzymes observed in aspirin-treated EG110 yeast cells indicates an aspirin-induced impairment of the carnitine shuttle pathway in these cells during growth in ethanol medium, as initially suggested by the microarray and qRT-PCR results.Similarly, aspirin decreased the overall carnitine acetyltransferase (CAT) enzyme activity in redox-compromised EG110, but not in wild-type EG103, yeast cells Table . Yeast C50, which would have interfered with the activity assay readings of peroxisomal Cit2 present in the cytosolic extracts.Likewise, the activity of peroxisomal citrate synthase Cit2) declined significantly in MnSOD-deficient EG110 yeast cells, but remained unaltered in wild-type EG103 yeast cells cultivated for 48 h in aspirin-treated ethanol medium . The ADH2-overexpressing strains EG110 pGPD-ADH2, and the respective wild-type EG103 pGPD-ADH2 control, were constructed by replacing the endogenous promoter of ADH2 with the constitutive GPD promoter.In view of the pronounced aspirin-induced suppression of GPD promoter.Immunoblot analysis of strains carrying HA fusion constructs of the Adh2 protein allowed us to compare the quantity of expressed Adh2 after growth of the cells in ethanol medium, in the absence and presence of aspirin. This assay indicated an aspirin-induced decrease of Adh2 expression in MnSOD-deficient EG110 cells Fig. . HoweverpGPD-ADH2 cells sustained a constant level of Adh2 activity even in the presence of aspirin, whereas the EG110 cells did not . The distinct response to aspirin of these two strains, described in past studies carried out in our laboratory20, was indeed again made immediately apparent in our transcriptome analysis, which showed that aspirin altered the gene expression profile of EG110 cells to a greater and significantly different extent than that of EG103 cells  cells that imparts very distinct gene expression profiles to these two strains  enzymes  was a key factor in averting aspirin-induced apoptosis21. Moreover, in the present study, the induced overexpression of active Adh2 , known to be a central factor underlying the survival, uncontrolled proliferation and progression of numerous types of cancer cells68. The importance of acetyl-CoA metabolism in cancer cells is further underscored by studies which show that the rate of synthesis of acetyl-CoA, mediated by the mammalian cytosolic enzyme acetyl-CoA synthetase (ACSS2), is significantly elevated in several malignant cell lines68 in order to satisfy their high energy requirements, particularly under conditions of metabolic stress.The suggested influence of aspirin on acetyl-CoA metabolic pathways in our redox-compromised yeast cells may provide the key for understanding the chemopreventive, antineoplastic effects of aspirin in early developing tumour cells, which also suffer increased oxidative stress and altered metabolism67. Likewise, targeted disruption of key carnitine shuttle enzymes mediating transport of acetyl-CoA to the cytosol of lymphoma cancer cells for fatty acid synthesis, was shown to cause a cytotoxic depletion of acetyl-CoA and growth inhibition in these cells69. In addition, cross-talk between acetylation and histone modifications strongly implicate acetyl-CoA in the regulation of chromatin acetylation and gene expression71. Hence, the strong association between aspirin and impairment of acetyl-CoA metabolism, highlighted in this study, merits further investigation in the context of aspirin’s anti-cancer properties.The strong reliance of cancer cells on metabolic pathways that supply acetyl-CoA, has recently raised interest in the potential use of antineoplastic agents targeting acetyl-CoA. For instance, studies have shown that targeted inhibition of ACSS2-mediated acetyl-CoA synthesis caused growth inhibition of cultured tumor cells and significantly reduced the growth and size of tumor xenografts in miceFarrugia et al., 2018 - Supplementary Information_ Aspirin impairs acetyl-coenzyme A metabolism in redox-compromised yeast cells


However, the mechanism by which UBIAD1 participates in tumorigenesis remains unknown. This study show that UBIAD1 interacts with H-Ras, retains H-Ras in the Golgi apparatus, prevents H-Ras trafficking from the Golgi apparatus to the plasma membrane, blocks the aberrant activation of Ras/MAPK signaling, and inhibits the proliferation of bladder cancer cells. In addition, GGPP was required to maintain the function of UBIAD1 in regulating the Ras/ERK signaling pathway. A Drosophila model was employed to confirm the function of UBIAD1/HEIX in vivo. The activation of Ras/ERK signaling at the plasma membrane induced melanotic masses in Drosophila larvae. Our study suggests that UBIAD1 serves as a tumor suppressor in cancer and tentatively reveals the underlying mechanism of melanotic mass formation in Drosophila.UbiA prenyltransferase domain-containing protein 1 (UBIAD1) plays a key role in biosynthesis of vitamin K One of the central paradoxes involving Ras signal transduction is how Ras, which acts as a simple binary switch, can generate many different biological outputs5. The intracellular trafficking cycle of Ras partially accounts for this phenomenon6. Ras interacts dynamically with specific microdomains of the plasma membrane and other internal cell membranes. These different membrane microenvironments modulate Ras signal output, highlighting the complex interplay between Ras location and function7. For example, Ras signaling in the plasma membrane is rapid and transient, whereas that in the Golgi apparatus is delayed and sustained8. Ras activates Raf-MEK-ERK in the plasma membrane and leads to cellular growth6. Cellular differentiation is induced when Ras activates Raf or PI3K in the Golgi apparatus10.Ras proteins are essential components of the intracellular signaling cascades that regulate various fundamental cellular activities such as proliferation, apoptosis, differentiation, and senescence6, is also common in thyroid cancer, salivary carcinoma11, epithelial myoepithelial cancer and kidney cancer12. After being synthesized in cytosolic ribosomes, H-Ras undergoes farnesylation at the cysteine of the C-terminal CVLS motif in the cytosol, followed by proteolysis of the VLS sequence in the endoplasmic reticulum (ER). However, as farnesylation of Ras provides only a weak signal for membrane interaction, another motif in the HVR (hypervariable region) of Ras is required for membrane association. Two palmitoylation groups at the cysteine, adjacent to the farnesylated cysteine of H-Ras, are the second membrane-anchoring signal. Palmitoylation primarily occurs at the Golgi surface, and palmitoylated H-Ras then aggregates to the plasma membrane via a secretory pathway for eventual insertion into the plasma membrane13. In addition, palmitoylated H-Ras undergoes dynamic de/re-acylation with depalmitoylation leading to detachment from the plasma membrane to facilitate transfer to the Golgi apparatus15. Bladder carcinoma with H-Ras mutation16 ranks ninth in cancer incidence worldwide17. Considering that H-Ras induces melanoma development in mice18 and melanotic spots appear in heix/ubiad1 mutant Drosophila19, we focused on establishing the relationship between UBIAD1 and H-Ras.H-Ras, which accounts for all Ras mutations in bladder carcinomaUBIAD1 , a cancer suppressor gene in bladder carcinoma21, encodes a class of UbiA prenyltransferases22 and participates in the regulation of cholesterol26. UBIAD1 is widely expressed in various human tissues20 and organelles, including the endothelial reticulum22, Golgi apparatus28, and mitochondria29. Interestingly, UBIAD1, with both side-chain cleavage and its prenylation activity, is the first enzyme responsible for human vitamin K biosynthesis30. Moreover, UBIAD1 is a nonmitochondrial CoQ10-forming enzyme with a specific cardiovascular antioxidant function via regulation of eNOS activity in zebrafish27. Given that UBIAD1 participates in various biological processes, its downregulation or mutation can induce the development of diseases such as cancer31, Schnyder crystalline corneal dystrophy (SCCD)32, Parkinson’s disease29, and cardiovascular disease27.35 and plays an important role in vascular cell calcification36. DNA methylation of ubiad1 has significant negative associations with EGFR/KRAS mutations in lung adenocarcinoma37. Furthermore, considering that UBIAD1 is downregulated in bladder and prostate carcinomas, and its overexpression inhibits tumor cell proliferation38. We previously reported that UBIAD1 knockdown activates the Ras/MAPK signaling pathway39.Recent research on this tumor suppressor show that UBIAD1 protects the cholesterol biosynthetic enzyme HMG-CoA reductase from degradationHere, we report that UBIAD1 interacts with H-Ras, increases the retention of H-Ras in the Golgi apparatus, inhibits the aberrant activation of Ras/ERK signaling at the plasma membrane and consequently suppresses the proliferation of bladder cancer cells.39, and UBIAD1 has inhibited the growth of bladder  in T24 cells  and UBIAD1. UBIAD1 inhibited both H-Ras-induced and H-RasG12V-induced p-ERK  Drosophila mutants . Neither heixk11403 nor heix1 express the HEIX protein29. These findings are consistent with a previous study reporting that heix regulates expression of yan, which is mediated by Ras19. Overexpression of the heix gene in heix mutants decreased phosphorylated ERK levels and led to the subsequent disappearance of melanotic masses , suggesting that melanotic mass results from abnormal activation of Ras/ERK signaling  was overexpressed in HEK293T cells, H-Ras was widely localized in the plasma membrane with little traces in the Golgi apparatus, which is consistent with previous reports42. However, when coexpressed with UBIAD1-EGFP in HEK293T and T24 cells, the localization of H-Ras in the Golgi apparatus significantly increased  in the Golgi apparatus.Considering that UBIAD1 is a Golgi-localized protein  and then in the Golgi apparatus43. However, when GFP-RBD and H-Ras were coexpressed with UBIAD1, UBIAD1 prevented the transport of H-Ras to the plasma membrane under short-term treatment with EGF   assay was performed to analyze the possible connection between the retention of H-Ras in the Golgi apparatus and the decrease in p-ERK. Under unstimulated conditions, GFP-RBD was not associated with any intracellular structures, including the plasma membraneEGF Fig. . UBIAD1 EGF Fig. . Interes44) Fig. . Ras inh44) Fig. . This reheix mutants was related to Ras signaling, an inhibition assay was performed, fewer traces of melanotic masses were detected in the heix mutants when treated with FTI-277, Salirasib, 2BP, tunicamycin  and coimmunoprecipitation (co-IP) assays were performed. BIFC signals were detected only in HEK293T cells cotransfected with UBIAD1 and H-Ras  assay was employed to detect this molecular interaction. Significant FRET signals were detected with 405-515 microscope channels when HEK293T cells were cotransfected with UBIAD1 and H-Ras Fig. . To deteing Fig. . Furthering Fig. . A smalling Fig. , and endvae Fig. . These d46. To assess the effect of H-Ras trafficking on the interaction between UBIAD1 and H-Ras, we performed knockdown and inhibition assays. We found that interaction between UBIAD1 and Ras decreased following knock down of FTNA (α-domain of FTase and GGTase) , and the terminal prenylated cysteine residue is methylated by isoprenylcysteine methyltransferase (Icmt) in the ER. Finally, the complex is palmitoylated by palmitoyltransferase in the Golgi apparatus and transported to the plasma membranese) Fig.  and Icmtse) Fig. , respectse) Fig. , indicatG12V-induced p-ERK in cells overexpressing H-Ras-CT  can inhibit the mevalonate pathway via 3-hydroxy-3-methylglutaryl-coenzyme A reductase, leading to a simultaneous decrease in the production of FPP (farnesyl pyrophosphate) and GGPP48. In our study, we found that statins abrogated the influence of UBIAD1 on the Ras/ERK signaling pathway , which synthesizes GGPP from FPP49. Thus, knockdown of GGPPS decreased interaction between UBIAD1 and H-Ras, which was abrogated by supplementary GGPP , which binds to the C-terminus of Ras and facilitates the transit of Ras proteins to either the Golgi apparatus or the recycling endosomes58. UBIAD1 inhibited H-Ras trafficking from the Golgi apparatus to the plasma membrane and inhibited the proliferation of bladder cancer cells. Ras translocates to the plasma membrane in the absence of UBIAD1, leading to the formation of cancer. Therefore, UBIAD1 has a similar function as PDEδ inhibitors or FKBP12 in regulating Ras trafficking between the plasma membrane and the Golgi apparatus (or ER). UBIAD1 may be a potential target for regulating Ras trafficking.Here, we found that UBIAD1 interacted with the C-terminus of H-Ras, which is consistent with the binding data for FKBP12 and PDEδ5. Ras sends different signal outputs in various regions such as the plasma membrane, Golgi apparatus, ER and mitochondria59. These events may occur through several factors, such as the different MAPK scaffolds employed by the plasma membrane and the Golgi apparatus3. Ras/ERK signaling activated by Ras in the plasma membrane induces cell growth, and Ras in the Golgi can regulate the Raf/ERK, PI3K/AKT/mTOR or PLC-γ-Ca2+ signaling pathway, leading to cell differentiation60. PAQR9/10 bound to H-Ras in the Golgi retains H-Ras in the Golgi and induces cell differentiation9. ENOS activates N-Ras in the Golgi and induces T-cell dependent apoptosis61. Additionally, FKBP12 promotes H-Ras trafficking from the plasma membrane to the Golgi, activates H-Ras in the Golgi, and induces cell differentiation under EGF treatment15. Our findings show that UBIAD1 prevents H-Ras trafficking from the Golgi to the plasma membrane. UBIAD1 is essential in embryonic mouse development30, and heix is specifically expressed in a small cluster of hemocytes within the Drosophila embryonic head mesoderm, a developmental zone of hemocytes19. These findings suggest a new function of the UBIAD1 protein in cell differentiation during biological development. UBIAD1 may regulate the balance between cell proliferation and differentiation by mediating plasma membrane- Golgi cycling of H-Ras.Ras is an important protein implicated in cell proliferation circuits, and can influence other cell circuits, such as motility, cytostasis, differentiation, and viability circuits20, prostate cancer21, and renal carcinoma31. UBIAD1 can inhibit the cell cycle and cell proliferation by regulating cholesterol and SXR target genes in the ER or mitochondria62. Here, the results demonstrate that UBIAD1 inhibits cell growth by regulating Ras/ERK signaling in the Golgi apparatus, suggesting that UBIAD1 is involved in the inhibition of cell proliferation. UBIAD1 is a membrane-associated protein with different functions in the ER and Golgi apparatus47, and GGPP is necessary during the transport of this protein from the ER to the Golgi apparatus63. When GGPP is insufficient, the transport of UBIAD1 to the ER under treatment with statins or GGPPS knockdown induces HMG-CoA reductase synthesis of GGPP and cholesterol, causing cell proliferation33. By contrast, transport of UBIAD1 to the Golgi apparatus in the presence of GGPP causes the protein to bind to H-Ras, which consequently prevents the trafficking of H-Ras from the Golgi apparatus to the plasma membrane and inhibits cell growth. UBIAD1 mutants lack the ability to use GGPP and are therefore located in the ER instead of the Golgi apparatus, inducing cholesterol generation and SCCD disease33. These data show that UBIAD1 mutation or wild-type UBIAD1 without GGPP are localized in the ER and both types lose their functionality, consistent with previous studies. Therefore, UBIAD1 is in a dynamic balance between the ER and the Golgi apparatus to regulate cell growth.UBIAD1 suppresses tumor growth in bladder cancer18, and a lack of heix can also cause melanotic mass formation in Drosophila larvae19. Therefore, H-Ras and UBIAD1 may be associated with melanoma formation. Our data show that large amounts of H-Ras aggregate in the plasma membrane and activate Ras/ERK signaling, causing melanotic mass formation under heix-deficient conditions. The mechanism responsible for melanotic mass formation in Drosophila may be similar to that in mice. Melanotic masses, the most obvious feature of heix mutation in Drosophila19, can be subdivided into melanotic nodules engaging the hemocyte-mediated encapsulation and melanizations that are not encapsulated by hemocytes64. Loss of function of heix or gain of function of Ras leads to an increased proliferation of hemocytes45. Hemocyte proliferation is closely correlated with the formation of melanotic mass, which can be attributed to an increase in the number of crystal cells associated with melanization66. Our study show that heix mutant leads to activation of Ras signaling on the plasma membrane. Therefore, the formation of melanotic mass in a heix mutant may result from Ras-induced proliferation of hemocytes. Activated Ras reportedly increases macropinocytosis and autophagy and directs glucose metabolism into hexosamine biosynthetic pathways by upregulating many key enzymes involved in glycolysis51. Therefore, UBIAD1/HEIX knockdown induces Ras/MAPK signaling and might reprogram metabolism, leading to tumorigenesis. In addition, it is reported that the melanotic mass formation in Drosophila is linked to a hemocyte-mediated immune response64. Previous studies report that loss of heix function not only activates the Ras signaling pathway but also leads to the activation of immune-related pathways 66. Therefore, besides the Ras pathway, the formation of melanotic mass in heix mutants may be influenced by immune-related pathways.The establishment of carcinoma is a complex process. Ectopic expression of H-Ras can induce melanoma in miceDrosophila. Our findings confirm the tumor suppressor function of UBIAD1 in cellular circuits in melanotic mass formation in Drosophila.In summary, UBIAD1 bind to H-Ras, regulates the H-Ras intracellular trafficking cycle, inhibits Ras/MAPK signaling, suppreses bladder cancer cell proliferation and plays important roles in melanotic mass formation in 2. Transient transfection of HEK293T and T24 cells was performed with LipofectamineTM 200028.Human bladder cancer cells T24 and human embryonic kidney cells HEK293T were obtained from American Type Culture Collection. HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (Hyclone) supplemented with 10% fetal bovine serum (FBS).T24 cells were cultured in minimum essential medium  supplemented with 10% FBS. All cultures were maintained at 37 °C in a humidified incubator with 5% CO67Total RNA was extracted from HEK293T cells with TRIzol reagent , and cDNA was synthesized using the first-strand cDNA synthesis kit . Real-time quantitative PCR was performed using the double-stranded DNA dye SYBR Green  to quantify the amount of gene expression. Gene expression assays were performed using procedures recommended by ABI Biosciences, and data were analyzed using ΔΔCt values. Primer pairs for UBIAD1 were used according to a previous study.The mRNAs levels were normalized to ACTIN.The primer pairs of ACTIN:(ACTIN-F:5'-CGCGAGAAGATGACCCAGAT-3'ACTIN-R:5'-GTACGGCCAGAGGCGTACAG-3').28. For construction of DsRed-H-Ras, full-length human H-Ras cDNA was amplified by PCR and cloned with DsRed-Monomer into the pcDNA3.1 vector. BFP-H-Ras was constructed by fusing H-Ras at the N-terminus of BFP into the pcDNA3.1 vector. GFP-H-Ras and its mutation were cloned into pEGFP-C1. Flag-H-Ras and its mutation were constructed by fusing the Flag tag to the N-terminus of H-Ras and inserting into pcDNA3.1. The plasmid 3Flag-UBIAD1 was constructed by fusing the 3Flag tag to the N-terminus of UBIAD1 and inserting into pcDNA3.1. pGFP-RBD was kindly provided by Dr. Mark R. Philips. Full-length human UBIAD1 and Apo E cDNA fragment were separately cloned into the pEGFP-N1 vector to create UBIAD1-EGFP and Apo E-EGFP. UBIAD1 was cloned into pcDNA3.1 to generate pcDNA3.1-UBIAD1. UBIAD1-BFP, Golgi-BFP and Apo E-BFP were constructed by fusing BFP with the C-terminus of UBIAD1 and Apo E and inserting into pcDNA3.1. All vectors for the BIFC assay were constructed using pcDNA3.1.The plasmid pOTB-UBIAD1 was purchased from Open Biosystems Inc. Enhanced green fluorescent protein (EGFP) vectors pEGFP-N1, pEGFP-C1, pCasper3-BG (TAGBFP-GFP), mammalian expression vector pcDNA3.1 and TAGBFP were obtained from Invitrogen. pDsRed-Golgi vector was obtained from Clontech, previously describedHEK293T cells were mounted onto the polylysine-slides and were 4% formaldehyde fixed (10 min) to permeabilize cells. Cells were incubated in 1% BSA (Bovine Serum Albumin)/10% normal goat serum in 0.1% PBS -Tween for 2 h to block non-specific protein-protein interactions. Cells were then incubated with primary antibodies overnight at 4 °C. After several washes with cold PBS, the slides were incubated with secondary antibodies for 2 h at room temperature. Fluorescence was observed by confocal microscopy  using 488 argon ion laser for GFP/488, a 543 He-Ne laser for RAF/549, and a 405 diode laser for BFP/DAPI. FLUOVIEW (Olympus) was used as the image acquisition software. Images were acquired, processed and analyzed with Image J .Drosophila were as follows: U0126 (10 μM), FTI-277 (10 μM), Salirasib (35 μM), 2BP (150 μM), tunicamycin (10 μM), and simvastatin (10 μM). FPP (#63250) and GGPP (#63330) were purchased from Cayman.Cells were starved for 6 h, cultured with DMEM and stimulated with 100 ng/ml EGF for different periods of time. The inhibitors U0126 , FTI-277 , Salirasib , 2BP , tunicamycin , and simvastatin  were dissolved in DMSO (Dimethyl sulfoxide). The working concentrations for cells and Drosophila were as follows: p-ERK (sc-7976) and ERK2 (sc-153) from Santa Cruz (US), pan-Ras (#3339) from Cell Signaling Technology (USA). HEIX antibodies were synthesized by GL Biochem , as previously described19.The following antibodies were used in this study: p-c-Raf (Ser259) (#9421), p-c-Raf (Ser338) (#9427), p-c-Raf (Ser289/296/301) (#9431), c-Raf (#9422), p-ERK1/2 (#4370), total-ERK1/2 (#4695), and caspase-3 (#9662) from Cell Signaling Technology (USA); anti-phospho-MEK (Ser218/222)/MEK2 (Ser222/226) (#2283379) from Millipore (USA); MEK1/2 (Ab-217/221) (#21203) antibody from Signalway Antibody (USA); Antibodies detecting actin (A01010-1) and GAPDH (A01020-1) from Abbkine (USA); Flag tag monoclonal antibody (A00187) from GenScript (China); GFP tag antibody (#66002-1) and HMGB1 (#10829-1) from proteintech (China); 488/549/645-conjugated secondary antibody from Abbkine (USA); HRP-conjugated secondary antibody from ABGENT (USA); UBIAD1 antibody (ab36832), Pan-Ras antibody (ab16907), and LC3B (AB192890) from Abcam (USA); rabbit IgG, mouse IgG from Immunoreagents (USA). Antibodies used for experiments with 39. In short, proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (12% gel) and transferred to PVDF (Polyvinylidene fluoride) membrane. The membranes were subsequently blocked with 5% fat-free milk dissolved in TBS  containing 0.1% Tween for 3 h at room temperature and then incubated overnight with primary antibodies at 4 °C. After incubation with horseradish peroxidase-conjugated secondary antibody, proteins were detected using enhanced chemiluminescence (ECL). Band intensity was quantified with Quantity One software.An immunoprecipitation assay was performed 48 h after cells were transfected with plasmids. Cells were lysed in lysis buffer (50 mM Tris-HCl (pH = 7.4), 150 mM NaCl, 0.5 mM EDTA and 0.1% NP-40, supplemented with protease inhibitor cocktail (CST #5871S) and phosphorylated protease inhibitor cocktail (BOSTER #AR119)), frozen and thawed once. Cell lysates and specific antibodies were incubated at 4 °C with continuous mixing overnight. Fifty microliters protein G-coupled beads  were prepared (according to the supplier’s instructions), and the mixture containing the pre-formed antibody-antigen complex was added to the beads. After 3 h of incubation at 4 °C, the supernatant was removed, and the beads were washed five times with cold PBS containing 0.1% Tween 20. Following the final wash, 60 μl sample buffer was added and the reaction was heated at 90 °C. The sample was detected by western blotting as previously described by Xia and collaborators26 5'-CACCGTAAGTGTTGACAATTACCGGTTCAAGAGACCGGTAATTGTCAACACTTACTTTTTTG-3'.shRNA vector-pGPU6/GFP/Neo was constructed by GenePharma . sh-UBIAD1:sh-NC: 5'-CACCGTTCTCCGAACGTGTCACGTCAAGAGATTAACGTGACACGTTCGGAGAATTTTTTG-3'.68 The shRNA vector into PGH1/RFP/Neo were constructed by GenePharma. 5'-CACCGATCCGGTGCCGCAGAATGATTCAAGAGATCATTCTGCGGCACCGGATTTTTTTG-3’. sh-NC: 5'-CACCGTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTTTG-3'.sh-FNTA:si-RNA oligonucleotides were constructed by GenePharma .39 5'-GUAAUUUGGUCAACACUUATT-3'si-UBIAD1:49 5'-GUCCCACUGAAGAAGAAUA-3'.si-GGPPS:69 5'-CCAUAGCUUAUAUUCUCAAdTdT-3'.si-Icmt:si-NC: 5'-UUCUCCGAACGUGUCACGUTT-3'.5 cells per well; after 24 h, the cells were transfected with plasmids. Cell viability was evaluated using the MTT  assay, according to the manufacturer’s protocol.T24 cells were plated in 96-well plates at a density of approximately 103 cells per well; after 24 h, the cells were transfected with plasmids. Cell proliferation was evaluated using the BrdU cell proliferation ELISA kit (colorimetric) , according to the manufacturer’s protocol.T24 cells were plated in 96-well plates at a density of approximately 1019. heixk11403/Df and heixk11403/heix1 are mutations of heix. For the P-element allele heixk11403, the insertion site is between bases 2L: 16299582 and 2L: 16299583 (transcription initiation area) and is expected to induce a non-expression (Bloomington stock 11031). For the ethylmethansulfonate (EMS) allele heix1, an AR144 mutation resulting from a G to A base change in the last residue of the only intron and is expected to result in an aberrant splicing event (Bloomington stock 3600). The deficiency allele Df(2 L)RA5/CyO (Bloomington stock 6915). The control is wild-type (w1118), and heixk11403/Df; tub P > heix is the rescue type.The stocks used in this study were previously describedDrosophila was prepared in a tube on the first day, and treatments were added to the food surface on the second day. Virgin flies were placed in the tube on the third day, and crossing was performed on the fourth day. The flies were removed from the tube 1 week later.Food for t-test, p < 0.05 was considered statistically significant. Each experiment was performed at least in triplicate.Results are presented as the mean ± standard deviation (SD). Statistical data comparisons among groups were performed using a non-parametric, Student’s supplementary figure


C. elegans, we identified crld-1 as a gene required for the synaptic expression of ionotropic acetylcholine receptors (AChR). We demonstrated that the CRLD-1A isoform is a membrane-associated ER-resident protein disulfide isomerase (PDI). It physically interacts with AChRs and promotes the assembly of AChR subunits in the ER. Mutations of Creld1, the human ortholog of crld-1a, are responsible for developmental cardiac defects. We showed that Creld1 knockdown in mouse muscle cells decreased surface expression of AChRs and that expression of mouse Creld1 in C. elegans rescued crld-1a mutant phenotypes. Altogether these results identify a novel and evolutionarily-conserved maturational enhancer of AChR biogenesis, which controls the abundance of functional receptors at the cell surface.The assembly of neurotransmitter receptors in the endoplasmic reticulum limits the number of receptors delivered to the plasma membrane, ultimately controlling neurotransmitter sensitivity and synaptic transfer function. In a forward genetic screen conducted in the nematode  The total amount or neurotransmitter receptors synthesized within a neuron or a muscle cell determines the size of the receptor pool that can be delivered to the plasma membrane and, ultimately, the responsiveness of the cell to specific transmitters. Assembly of multiple subunits into mature receptors in the ER seems to be an inefficient and limiting step in the synthesis of ligand-gated ion channels (LGCI) belonging to the Cys-loop superfamily of receptors .A, glycine and serotonin 5-HT3 receptors. They are made of five identical or homologous subunits arranged around a fivefold pseudo-symmetrical axis , also includes GABAcal axis . Each sucal axis . In musccal axis . Similareceptors .4β2 AChRs from the ER were associated with amyotrophic lateral sclerosis  and death of wild-type worms at high concentrations . Creld1 is widely expressed in humans, with the highest levels in adult heart, brain and skeletal muscle  transcripts revealed that cryptic splice donor sites present in the Mos1 kr133 transposon were used at low frequency to generate in-frame mRNAs . None of the crld-1 mutants exhibited a grossly abnormal phenotype. Specifically, locomotion remained coordinated and only a slight decrease of the thrashing frequency in liquid could be detected in tm3993 mutants but not in Mos1 alleles. The most dramatic phenotype was a decreased sensitivity to levamisole since almost 100% of the crld-1 mutant animals fully adapted overnight to 1 mM levamisole while all the wild-type animals were paralyzed  had wild-type sensitivity to levamisole and provided a means to visualize the expression pattern of both CRLD-1 isoforms (9a and −9b of crld-1(kr298::gfp) with a stop codon in order to suppress the expression of crld-1a and crld-1b isoforms, respectively (crld-1a-specific mutants were as resistant to levamisole as crld-1(tm3993) while crld-1b-specific mutants were indistinguishable from the wild type  animals expressing both crld-1a and crld-1b isoforms tagged with gfp displayed a combination of the two patterns  mutants. In contrast, we observed an obvious decrease of synaptic L-AChRs stained by anti-UNC-38 antibodies in crld-1(tm3993) as compared to the wild type (crld-1 mutant as compared to the wild type (crld-1(tm3993) mutants compared to the wild type  mutants and the wild type  mutants and the wild type (crld-1(tm3993) mutants  mutants might result from decreased synthesis or from intracellular retention of receptors. To distinguish between these hypotheses we quantified the overall amount of UNC-29 L-AChR subunit by western blot analysis. UNC-29 expression was decreased by approximately 50% in crld-1(tm3993) mutants as compared to the wild type (crld-1(tm3993) mutants (The reduction of L-AChR at the NMJ of ild type . To dete mutants .unc-63(kr13) null mutant background: in the absence of the obligatory AChR subunit UNC-63, the remaining unassembled subunits, such as UNC-29, are retained in the ER and can be readily detected by western blot analysis  and N-glycosidase F (PNGaseF)  mutant background. By contrast, the digestion profile of UNC-29 in crld-1(tm3993) was similar to the wild type, suggesting that the subunits that were not degraded in crld-1(tm3993) were properly assembled into functional receptors that could traffic to the plasma membrane (To confirm that remaining receptors in PNGaseF) . Nascentmembrane .A receptor  or L-AChR assembly intermediates.C. elegans CRLD-1 identified a conserved C27XXC30 motif in the N-terminal region of the CRLD-1 WE domain (It was recently shown that human CRELD2 is a putative protein disulphide isomerase (PDI) . PDIs caE domain .gfp-crld-1 knock-in  (wild type) and gfp-crld-1(kr302) (C30A mutant) were separated by SDS-PAGE under reducing and non-reducing conditions. GFP-CRLD-1 was revealed by western blot analysis using an anti-GFP antibody. Under non-reducing conditions we detected high molecular weight species containing the mutated GFP-CRLD-1(C30A) that were absent in the non-mutated GFP-CRLD-1  null mutant , receptors were then solubilized, pulled-down with streptavidin and quantified by western blot. As compared to control cells (shScramble), shCreld1 caused a 50% decrease of AChR expression and further decreased the ratio of surface vs total AChR by 50% (AChRα subunit and Myogenin (marker of differentiation) were not affected by Creld1 downregulation  is cell-autonomously required in both nematode and mammalian muscle cells to promote the surface expression of AChRs. We propose that this protein could act in the ER as a maturational enhancer to stabilize unassembled AChR subunits and to promote AChR assembly.CRLD-1 was identified in a genetic screen for mutants partially resistant to the cholinergic agonist levamisole. This gene is evolutionarily conserved and our results show that the membrane-associated isoform of CRLD-1 (CRLD1-A in C. elegans suggests that CRLD-1A is an intrinsic ER-membrane protein with the C- and N-termini facing the ER lumen.The transmembrane isoform CRLD-1A (ortholog of vertebrate CRELD1) contains two conserved transmembrane regions located at the C-terminus of the protein. Bioinformatic analysis of human CRELD1 suggests that both N- and C-termini reside in the extracellular spaces with a short intervening cytoplasmic loop . In contCreld1 cDNA rescues C. elegans crld-1 mutants, it seems reasonable to propose that both mouse and nematode proteins have the same topology.First, CRLD-1A and CRLD-1B isoforms are identical except for their short C-terminal regions . Bioinformatic analyses identify a signal peptide at the N-terminus of CRLD-1, which predicts that the N-terminal region of CRLD-1 is translocated into the ER lumen. This is fully consistent with the localization of CRLD-1B, which behaves as a luminal ER protein. Second, over the course of our experiments, we overexpressed in muscle cells CRLD-1A fused to GFP at its N-terminus and we detected fluorescence at the plasma membrane of muscle cells, probably because some protein could escape the ER-retention machinery. In these transgenic worms, we injected fluorescently-labeled anti-GFP antibodies into the pseudo-coelomic cavity, a means to label cell-surface exposed epitopes  and we cCreld2 , unc-63(kr13); LG IV: crld-1.eviously  and keptStrains, expression constructs, transgenic animals and generation of knock-in worms are listed and described below.The following mutant alleles and transgenes were used in this study:unc-63(kr13), unc-29(x29), unc-29(kr208::tagRFP) ;LGI: unc-49(e407); unc-49(kr296::tagRFP)LGIII: creld-1(kr132::Mos1), crld-1(kr133::Mos1), crld-1(tm3993); crld-1(kr297::HySOG), crld-1(kr298::GFP), crld-1(kr302::GFP-C30A), crld-1b(kr303::GFP), crld-1a(kr308::GFP);LGIV: acr-16(ok789).LGV: The following transgenic lines were created for this study:crld-1(tm3993): krEx870, krEx871.Extrachromosomal array in crld-1(kr297) : krEx1277.Extrachromosomal array in in crld-1(kr132 ::Mos1): krEx456.Extrachromosomal array crld-1b(kr303::GFP): krEx1245 , krEx1250 .Extrachromosomal array in crld-1a(kr308::GFP): krEx1246 , krEx1249 .Extrachromosomal array in C. elegans germline transformation:Transformation was performed by microinjection of DNA mixture in the gonad of young adults. The total DNA concentration of the injection mix was normalized at 100 ng/μL using 1kb + ladder (Invitrogen). The following plasmids were used for Pmyo-3::crld-1a cDNApTB205: Pmyo-3::crld-1b cDNApTB206: crld-1 and upstream regulatory regions fused to SL2-GFPpTB208: 4,6 kb genomic fragment containing Pmyo-3::RFP::MANS (pMR61: FP::MANS Pmyo-3::RFP::KDEL (pMR68: FP::KDEL Pmyo-3::mouse-creld-1 cDNA.pMD20: gfp-crld-1(kr298) knock-in was generated using the MegaTIC technique, this protocol consists of 2 steps .The final the crld-1(kr297) molecular null allele was generated by using the MosTIC technique as previously described  and negative  selection markers flanked by two meganuclease I-SceI target sites. The pMD1 vector was injected as a rescue template into a strain containing the kr133 Mos1 insertion in the fourth exon of crld-1 gene. In pMD1, a 1,5-kilobase (kb) left crld-1 homology sequence and a 4 kb right homology sequence flank the HySOG cassette. kr297 knock-in allele was identified using positive selection of worms containing the HySOG cassette, therefore selecting worms resistant to hygromycin B.In the first step, escribed . 49 nuclcrld-1 gene. In pHZ34, a 1,5-kilobase (kb) left crld-1 homology sequence and a 4 kb right homology sequence flank the gfp. Gfp-crld-1(kr298) knock-ins were identified based on their resistance to blue light illumination, followed by PCR analysis.In the second step, the HySOG cassette was excised by meganuclease-induced chromosomal breaks on each I-Sce-I site in the presence of pHZ34 as a repair template. pHZ34 contains gfp fused to the 5’ of the gfp-crld-1(kr302) knock-in containing the C30A point mutation was generated using a combination of Co-CRISPR and MegaTIC techniques. The starting point was the excision of the HySOG cassette from the crld-1(kr297) by using sgRNAs against each I-Sce-I site and in presence of the pMD3 repair template. pMD3 contains gfp fused to the 5’ of the crld-1 gene and the mutation TGC >GCT (C30A point mutation). In pMD3, a 1,5-kilobase (kb) left crld-1 homology sequence and a 4 kb right homology sequence flank the gfp. We chose dpy-10 as a co-conversion marker to introduce a dominant mutation causing a visible Rol/Dpy phenotype(The phenotype. Rol/Dpygfp-crld-1(kr298) knock-in was injected with sgRNAs against the splicing acceptor site of either exon 9a or exon 9b of crld-1 gene. Linear repair templates with short (≈30–40 bases) homology arms (gfp-crld-1(kr298). The linear repair templates contained PmeI restriction site followed by a STOP codon in place of the AG splicing acceptor site of either exon 9a or exon 9b. Dpy-10 was used as a co-conversion marker to introduce a dominant mutation causing a visible Rol/Dpy phenotype  flanked on each side by I-SceI sites, was inserted in pHZ34 vector between PstI and Bsp1407I sites. 763 nucleotides, encompassing the first exon of crld-1 gene fused to the gfp gene, were removed. Different fragments were assembled using isothermal assembly (pMD1: assembly .crld-1 gene.pMD3: this plasmid was created on the basis of pHZ34. Using Gibson cloning the C30A point mutation (TGC >GCT) was introduced in the sequence of C. elegans U6 promoter and 3' UTR (based on pMD5 (1 st I-SceI sgRNA): This vector was created on the basis of pPT02. The pPTbased on  and two pMD7 (2nd I-SceI sgRNA): This vector was created on the basis of pPT02. The protospacer sequence was inserted between the U6 promoter and the sgRNA scaffold, using PmeI and SexAI sites of pPT02. The protospacer contained the 3’ I-Sce-I site flanking the HySOG cassette in pMD1 and was synthetized by Sigma: AATTGCAAATCTAAATGTTTgAGGGATAACAGGGTAATCGCGTTTTAGAGCTAGAAATAGCdpy-10 sgRNA): This vector was created on the basis of pPT02. The protospacer sequence was inserted between the U6 promoter and the sgRNA scaffold, using PmeI and SexAI sites of pPT02. The protospacer was synthetized by Sigma: AATTGCAAATCTAAATGTTTgCTACCATAGGCACCACGAGGTTTTAGAGCTAGAAATAGCpMD8 (pMD10 (exon9a sgRNA): this vector was created on the basis of pPT02. The protospacer sequence was inserted between the U6 promoter and the sgRNA scaffold, using PmeI and SexAI sites of pPT02. The protospacer was synthetized by Sigma:AATTGCAAATCTAAATGTTTGATCAGGAGATGCTGAACCAGTTTTAGAGCTAGAAATAGC The linear templates used as repair template contained PmeI restriction site (GTTTAAC) followed by a STOP codon (TAA) in place of the AG splicing acceptor site of exon 9a and was also synthetized by Sigma:CACCCAGTTCCAATTTCCTCTATTCACCATGGTTCGTTTAAACTAACATCTCCTGATCGCCCGTTCATGCCAATCGACCAGCpMD11 (exon9b sgRNA): this vector was created on the basis of pPT02. The protospacer sequence was inserted between the U6 promoter and the sgRNA scaffold, using PmeI and SexAI sites of pPT02. The protospacer was synthetized by Sigma:GCTATTTCTAGCTCTAAAACTTGCTTTCAAGGCTGCAAATcAAACATTTAGATTTGCAATT The linear templates used as repair template contained PmeI restriction site (GTTTAAC) followed by a STOP codon (TAA) in place of the AG splicing acceptor site of exon 9b and was also synthetized by Sigma:CAATAAAATGTAGAATATTTTCATTTTTTCAAAATTTGCGTTTAAACTAACCTTGAAAGCAACGGAACAGCAAGCTCATGAAGATGEscherichia coli. Young adult worms were put on plates containing levamisole, animals were left overnight at 20°C, and paralyzed animals were then scored.Assays for levamisole sensitivity after overnight exposure were performed as previously described in . TetramiC. elegans, total RNA was isolated from a synchronized population of worms using the RNeasy Kit (Qiagen) according to the manufacturer’s instructions. In C2C12, myoblasts were cultured in 35 mm dishes and differentiated for 6 days before total RNA extraction using Trizol reagent (ThermoFisher). All samples were treated with DNase (Fermentas). First-strand cDNA was synthesized from 200 ng of total RNA using an oligo(dT) primer and a Moloney murine leukemia virus reverse transcriptase (Fermentas) at 42°C for 1 hr. Quantitative PCR was performed using LightCycler 480 SYBR Green I Master (Roche). A relative quantification model was used to evaluate the relative expression ratio of the target genes and RNA levels were normalized to three housekeeping genes ,for mouse RNAs).Quantitative Real-Time PCR assays were performed as previously described . In C. eElectrophysiological methods were performed as previously described by  and Ric.Worms were prepared by the freeze-crack method described previously . MethanoC2C12 cell lines were differentiated in myotubes and RT-PCR with primers specific for murine muscle was performed for cell identity confirmation. The cells were subjected to MycoAlert Mycoplasma detection kit from Lonza (LT07-318). C2C12 myoblasts were plated on Matrigel (Corning)-coated 35 mm dishes and grown in Hyclone DMEM  containing 4.5 g/l glucose, supplemented with 15% FBS (PAA) and penicillin/streptomycin. Cells were maintained at 37°C in a saturated humidity atmosphere containing 5% CO2. For the differentiation of C2C12 myoblasts, fetal bovine serum was replaced by 2% horse serum (Biowest) when myoblasts reached 70–80% confluence. Cells were differentiated for 6 days.Sh-Creld1 and control myoblast lines were obtained by selecting puromycin-resistant clones of C2C12 myoblasts, respectively transfected with shRNA clone set against mouse Creld1 or scrambled control clone (GeneCopoeia).DC Protein Assay according to manufacturer instructions (Bio-Rad). 30 μg of sample were boiled in 40 μl sample buffer and were loaded on 4‐12% pre‐cast gel (Biorad) and fast‐transferred into nitrocellulose membrane (Bio-Rad). Membrane was blocked with blocking buffer . Incubation with primary antibodies was done overnight in blocking buffer at 4 C. After three washes with PBS‐ Tween 0.002%, membranes were incubated with secondary antibodies coupled with HRP for 1 hr at room temperature. Proteins were visualized using LumiLight reagents (Roche).C2C12 cells were lysed in RIPA buffer (Abcam) supplemented with Complete protease inhibitor cocktail (Roche) and passed through a needle (Qiagen) to disrupt DNA. Protein concentration was measured with a Western blot membranes were probed with rabbit antibody anti-Creld1 , mouse commercial antibody anti-αAchR , mouse commercial anti-GAPDH , and secondary HRP-conjugated goat anti-rabbit or goat anti-mouse antibody .For surface labeling of AChR, C2C12 cells were incubated with 200 nM αBT-biotin (Invitrogen) in PBS for 30 min on ice. Cells were intensively washed in PBS after αBT-biotin and lysed in RIPA lysis buffer (as described above). Streptavidin-agarose  was used to recover biotinylated proteins. Quantitation of the Western blots was done by using the ChemiDoc MP Imaging System (Bio-Rad).C. elegans extracts are described in  β-mercaptoethanol, boiled at 90°C for 10 min and centrifuged for 5 min at 15,700 × g. For glycosylation profiles, samples were treated with denaturation buffer for 10 min at 90°C, incubated with endoglycosidase H  or PNGaseF (New England Biolabs) for 1 hr at 37°C, and subsequently treated with Laemmli buffer as described above. Membranes were imaged with a LAS4000  luminescence detector and band intensity-quantified with ImageJ software.Western blot membranes were probed with affinity-purified rabbit antibody anti-UNC-29 , mouse commercial antibody anti-TUBULIN , mouse commercial anti-GFP , and secondary HRP-conjugated goat anti-rabbit or goat anti-mouse antibody  and revealed with LumiLight reagents (Roche).A mixed stage population of worms (5 mL) was frozen at −80°C until use. For extraction, worm pellets were ground under liquid nitrogen and thawed in an equal volume of ice-cold worm lysis buffer  in 10 mL) . The susWorm extracts were prepared as described in the protocol of co-immunoprecipitation with the exception that 200 mM NEM  were added to the WLB buffer during protein extraction of samples in order to assay under not reducing conditions. Moreover sample tested under not reducing conditions were eluted in Laemmli buffer without beta-mercaptoethanol.Animals were mounted on 2% agarose pads, anaesthetized with 5 ml of M9 buffer containing 100 mM sodium azide and examined with either a Leica 5000B microscope equipped with a spinning disk CSU10 (Yokogawa) and a Coolsnap HQ2 camera, or a Nikon Eclipse Ti equipped with a spinning disk CSUX1-A1 (Yokogawa) and an Evolve EMCCD camera. Image analysis was performed with ImageJ.AR fluorescence, and L-AChR fluorescence in living worms, young adult animals were mounted on 2% agarose pads and immobilized using polybead microspheres  in M9 buffer. Quantification of synaptic GABAAR or L-AChR was achieved as described previously . The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Thank you for submitting your article "CRELD1 is an evolutionarily-conserved maturational enhancer of ionotropic acetylcholine" for consideration by C. elegans and also in mammals. The study is technically sound and there are no doubts about the quality of the results. The work shows an interesting new aspect of AChR biogenesis and the involvement of a new factor. Apart from some requested editorial changes (listed below), there were nevertheless three experimental aspects that the reviewers and editor suggest require further elaboration.The reviewers and editor appreciated the identification and phenotypic characterization of a novel factor regulating the biogenesis of nicotinic acetylcholine receptors (AChRs) in Essential revisions:crld-1 with unc-74. Because crld-1 mutants have a partial phenotype, there may be other players as well. An obvious candidate is UNC-74, which is a PDI as well, mutation of which eliminates all AChR biogenesis. This protein has been shown by the authors to be essential for expression of AChRs in a heterologous system (Xenopus oocytes) but has never been properly characterized in C. elegans. It is thus surprising that the authors do not analyze UNC-74 along with CRLD-1, as it could be that CRLD-1 shows a partial phenotype simply because the main player is UNC-74 (in whatever precise role in protein reduction/cysteine bridge formation is required during AChR biogenesis). Can the authors provide information about the expression and localization pattern of unc-74? And compare the effects of its removal on the effect of crld-1 removal?1) Exploring the regulation of and on reduced function that contributes to the electrophysiologically measured (reduced) currents? Are there ways to quantify expression levels via the fluorescent reporter tags?2) Figure 3A, C: The data presented do not exclude that AChR functional properties are altered and thus levamisole responses are different – while this may not have to do with a reduced expression level. The immunostaining suggests that, but from a single image, the reader cannot fully appreciate the differences between the genotypes. Can the authors exclude an effect that is both based on mislocalization 3) Figure 5C: The PDI assay is rather indirect. It provides reasonably good indication that CRLD-1 is a PDI, but what is the nature of the bands in the non-reduced version of the gel in the C30A variant? If the hypothesis is true, it should contain levamisole resisitant subunits. This could possibly be tested with the approach indicated in Figure 4, coIP of UNC-29::RFP with CRLD-1::GFP and specific detection in Western blot. If the UNC-29::RFP protein is part of the cysteine-linked species the authors may be able to detect RFP in the high-molecular weight bands. Or, directly probe their gel with anti-UNC-29 or anti-UNC-38 antibodies.crld-1 are based on their different localizations in the ER, but this is not based on more direct data. Also, notions of Creld1 possibly affecting nicotine addiction should include a reference to Hosur et al. (2009).– Discussion section: This section is lengthy and extends to areas not well touched by the content of the paper. Thus, some things appear overstated and could be toned down. E.g. the Discussion section speculates about how CRLD-1 helps AChR biogenesis, but not much direct data supporting functions as a chaperone are provided. Different functions of the two isoforms/splice variants of crld-1 confers partial resistance to the cholinergic agonist levamisole” 'these data demonstrate' or 'this data demonstrates'._ Subsection “Disruption of the evolutionarily conserved gene – Subsection “CRLD-1A and CRLD-1B are ubiquitously expressed and localize to the ER”: 'in most, if not all…'.– Subsection “CRELD1 and CRELD2 are conserved proteins controlling different processes in the ER”: Notion of differential levamisole sensitivity of specific mutants. I did not get what the authors refer to: Taking out A isoform eliminates levamisole sensitivity and taking out B isoform leaves it completely intact. So, it is an all-or-none requirement for A isoform, in all alleles affecting this isoform, nothing in-between/differential.– Figure 2. Scale bars are confusing and incomplete.crld-1 looks significant and could be interesting.– Figure 3E. The increased nicotine response in – None of the three words, protein disulphide isomerase, are proper nouns. That the acronym PDI is capitalized does not require capitalization of the full phrase.– "their very short C-terminal regions." Please provide exact length? Essential revisions:1) Exploring the regulation of crld-1 with unc-74. Because crld-1 mutants have a partial phenotype, there may be other players as well. An obvious candidate is UNC-74, which is a PDI as well, mutation of which eliminates all AChR biogenesis. This protein has been shown by the authors to be essential for expression of AChRs in a heterologous system (Xenopus oocytes) but has never been properly characterized in C. elegans. It is thus surprising that the authors do not analyze UNC-74 along with CRLD-1, as it could be that CRLD-1 shows a partial phenotype simply because the main player is UNC-74 (in whatever precise role in protein reduction/cysteine bridge formation is required during AChR biogenesis). Can the authors provide information about the expression and localization pattern of unc-74? And compare the effects of its removal on the effect of crld-1 removal?unc-74 mutants were isolated by Jim Lewis based on complete insensitivity to levamisole . unc-74 encodes a putative PDI , which has been analyzed by the Jorgensen's lab for several years. They demonstrated that no L-AChR is left in unc-74(0), and that UNC-74 is an ER protein that works cell autonomously to synthesize L-AChRs in muscle cells . A manuscript containing this data is in preparation by the Jorgensen's lab. Because no L-AChR is left in the unc-74(0) mutant, the unc-74(0); crld-1a(0) double mutant will not bring additional information on CRLD-1A function.2) Figure 3A, C: The date presented do not exclude that AChR functional properties are altered and thus levamisole responses are different – while this may not have to do with a reduced expression level. The immunostaining suggests that, but from a single image, the reader cannot fully appreciate the differences between the genotypes. Can the authors exclude an effect that is both based on mislocalization and on reduced function that contributes to the electrophysiologically measured (reduced) currents? Are there ways to quantify expression levels via the fluorescent reporter tags?A receptors at synapses using knock-in strains expressing fluorescently-tagged UNC-29 and UNC-49, respectively. We found that the fluorescence intensity of L-AChRs present at the ventral nerve cord was decreased by 85% in crld-1 mutant as compared to the wild type, while GABAR content was unchanged. Therefore, the reduction of the electrophysiologically measured currents in crld-1 mutants can be explained solely by the reduction of L-AChRs present at NMJs and does not imply an additional impairment of L-AChR function.As requested, we quantified L-AChR and GABAThese results are mentioned in the text (subsection “CRLD-1 is required for cell surface expression of L-AChRs”) and provided in Figure 3F-I.3) Figure 5C: The PDI assay is rather indirect. It provides reasonably good indication that CRLD-1 is a PDI, but what is the nature of the bands in the non-reduced version of the gel in the C30A variant? If the hypothesis is true, it should contain levamisole resistant subunits. This could possibly be tested with the approach indicated in Figure 4, coIP of UNC-29::RFP with CRLD-1::GFP and specific detection in Western blot: If the UNC-29::RFP protein is part of the cysteine-linked species the authors may be able to detect RFP in the high-molecular weight bands. Or, directly probe their gel with anti-UNC-29 or anti-UNC-38 antibodies.unc-29::RFP; crld-1::GFP double knock-in strain to immunoprecipitate CRLD-1::GFP (WT and C30A) and then detect UNC-29::RFP. In one experiment out of three, we were indeed able to detect RFP in the high molecular weight bands under non-reducing conditions using the C30A variant .This part of the Discussion section has been significantly shortened and the reference was included in the text.– Subsection “Disruption of the evolutionarily conserved gene crld-1 confers partial resistance to the cholinergic agonist levamisole”: 'these data demonstrate' or 'this data demonstrates'This was corrected.– Subsection “CRLD-1A and CRLD-1B are ubiquitously expressed and localize to the ER”: 'in most, if not all…'This was corrected.– Subsection “CRELD1 and CRELD2 are conserved proteins controlling different processes in the ER”: Notion of differential levamisole sensitivity of specific mutants. I did not get what the authors refer to: Taking out A isoform eliminates levamisole sensitivity and taking out B isoform leaves it completely intact. So, it is an all-or-none requirement for A isoform, in all alleles affecting this isoform, nothing in-between/differential."Differential sensitivity" was removed from the text– Figure 2. Scale bars are confusing and incomplete.The legend of Figure 2 was clarified, accordingly.– Figure 3E. The increased nicotine response in crld-1 looks significant and could be interesting.crld-1 mutants, this response is quite variable and there is no statistically significant difference between the crld-1 mutant and the wild type. The main point we get from this set of data is that crld-1 disruption does not cause a decrease of N-AChRs, as opposed to L-AChRs.Although we agree that it might be interesting if nicotine response would be increased in – None of the three words, protein disulphide isomerase, are proper nouns. That the acronym PDI is capitalized does not require capitalization of the full phrase.This was corrected.– "their very short C-terminal regions." Please provide exact length?The exact length of the 2 isoform C-terminal regions is now provided in the text (subsection “CRLD-1A membrane topology”).


To the Editor:www.jacionline.org). Serum reconstitution with proximal AP components suggested a factor D (FD) deficiency (A). Sanger sequencing of CFD identified a rare homozygous missense mutation (c.602G>C) in exon 4 in the patient (II-1) and sibling (II-2), resulting in an arginine to proline substitution (p. R176P) . This genotype cosegregated with an alternative pathway hemolytic activity–null phenotype, as the parents, both heterozygotes, had normal alternative pathway hemolytic activity (B). In contrast to previously confirmed FD deficiencies,B). Meanwhile, identical circular dichroism spectra and melting curves of recombinant wild-type (WT) and R176P FD precluded gross changes in FD structure or stability, suggesting a functional deficiency . We assessed the cleavage of C3b-bound factor B (FB) by recombinant WT and mutant FD . WT FD could cleave C3b-bound FB to produce fragments Bb and Ba. Conversely, R176P FD demonstrated diminished in vitro catalytic activity at all concentrations and had negligible activity at physiological concentration (0.04 μmol/L) . Reconstitution of FD-depleted serum with R176P FD also demonstrated impaired AP-mediated hemolysis (see E).We investigated an alternative complement pathway (AP) deficiency in a patient with absent alternative pathway hemolytic activity but normal classical pathway hemolytic activity recovering from invasive meningococcal infection .C). In mutant FD, we observed a rearrangement of the exosite loop 155-161 within 50 nanoseconds of simulation (A). This was unexpected because loop 155-161 was not in direct contact with residue 176. Average structures generated from the final 50 nanoseconds of simulation for WT and mutant FD (R176P and R176A) demonstrated that key FB-binding residues Asp161 and Arg157 were shifted by 4.3 Å and 1.9 Å, respectively (Cα average position) (B). Superimposing these MD average structures onto the crystal structure of the C3bB-D complex revealed that Asp161 and Arg157 assumed a conformation that no longer supported binding due to loss of shape and charge complementarity to the FB surface (C). The other 3 exosite loops retained their binding-competent conformations. After assuming the new conformation, exosite loop 155-161 demonstrated higher conformational mobility (root mean square fluctuation) relative to WT (D and E). In contrast, the mobility of loops containing catalytic residues His41 and Asp89 decreased in the mutants. Using the distance between His41 and Ser183 during MD simulations as a proxy for the active site conformation, we observed that WT could sample the short distance necessary for a catalytically active conformation (D). Conversely, in both mutant simulations, the distance remained larger, consistent with His41 pointing away from the active site. Therefore, in addition to disruption of key FB-binding residues, mutations R176P and R176A appear to stabilize the self-inhibited conformation of free FD.FD's serine protease activity depends on obligatory binding to the C3bB complex via 4 exosite loops . This leads to rearrangement of the self-inhibitory loop (199-202), allowing realignment of His41 and Asp89 with Ser183 to form the active catalytic triad  , B. Supe surface , C. The ve to WT , D and Eormation , D. ConvE). In contrast, R176P/S183A FD lacked any detectable binding (F). Consistent with the stochastic transitions of free WT FD to the active conformation observed in the MD simulation, FD has a low level of esterolytic activity toward a small synthetic substrate, Z-Lys-SBzl (D). Surprisingly, R176P FD demonstrated a loss of esterolytic activity similar to the active site mutant, S183A (see G).To assess the binding of FD to C3bB, we used surface plasmon resonance. Coinjection of catalytically inactive FD (WT/S183A) with FB demonstrated a dose-dependent increase in binding to C3b and complex formation , E. In c binding , F. ConsLys-SBzl , D. Surp183A see , G.www.jacionline.org). Meanwhile, previously confirmed deficiencies of activating complement serine proteases have all resulted in low or absent gene product. We have identified a unique deficiency: R176P FD is fully expressed and stable, but enzymatically inert, constituting a functional or type III deficiency. Recent preclinical evidencewww.jacionline.org).Deficiency of properdin, the most common AP deficiency, can result from absent (type I), low (type II), or normal but nonfunctioning (type III) protein levels .Our acknowledgments can be found in this article's Online Repository (at


Overexpression of miR-150 significantly decreased Notch3 expression in ADSCs compared with the control groups. Furthermore, Notch3 inhibition promoted the adipogenic differentiation in ADSCs. miR-150 also suppressed proliferation potential and the expression of Nanog in ADSCs. In summary, this study demonstrates, for the first time, that miR-150 promotes adipogenic differentiation and inhibits proliferation of ADSCs. miR-150 regulates adipogenic differentiation of ADSCs, likely mediated by the downregulation of Notch3.MicroRNAs (miRNAs) influence stem cell functions, including mobilization, proliferation, and differentiation. miR-150 is abundantly expressed in monocytes. Knockdown of miR-150 promotes bone marrow stem cell migration. The role of miR-150 in adipose-derived stem cells (ADSCs) is unclear. In this study, the effects of miR-150 on adipogenic differentiation and proliferation of ADSCs were investigated. ADSCs were isolated from the inguinal adipose tissue of wild-type (WT) and miR-150 knockout (KO) mice and were induced for adipogenic differentiation. The miR-150 level was detected by real-time PCR. ADSCs were transfected by miR-150 or small-interfering RNA (siRNA) of Notch3. MTT assay and colony formation assay were performed in miR-150 knockdown and control ADSCs. Real-time PCR showed that miR-150 was expressed in ADSCs. miR-150 knockdown significantly decreased the capacity of adipogenic differentiation of ADSCs, as compared with their counterparts from WT mice. It is intriguing that the overexpression of miR-150 significantly increased C/EBP ADSCs are a population of mesenchymal stem cells found in adipose tissue and are readily accessible from subcutaneous liposuction. ADSCs can self-renew and can differentiate into a number of cell types, which make them a viable option in regenerative medicine. The investigations show that ADSCs are a promising cell source for adipose tissue engineering as they can be differentiated into adipocytes . UnderstmiRNAs play an important role in the posttranscriptional regulation of target mRNA in a range of biological processes, including maintenance of stemness and modulation of mobilization, proliferation, and differentiation of ADSCs . Recent The Notch family members are highly conserved and have the effect of regulating the differentiation and proliferation of stem cells and progenitor cells . InhibitIn the present study, we investigated the mechanism of miR-150 in adipogenic differentiation of ADSCs. We demonstrated for the first time that miR-150 played an important role in ADSC differentiation towards the adipose by targeting Notch3.2 humidified atmosphere at 37°C. The third-passage cells were identified with flow cytometry. Human ADSCs (hADSCs) were kindly provided by Stem Cell Bank, Chinese Academy of Sciences. The hADSCs were harvested from a female donor using lipoaspirate, and the cells were expanded for five passages prior to cryopreservation and shipment. hADSCs were cultured under the same conditions as the ADSCs from mouse.ADSCs were isolated from the inguinal white adipose tissue from miR-150 KO mouse in a C57BL/6 background and wild-type (WT) C57BL/6 mouse (4-6 weeks) according to a published protocol from our laboratory . The ADSμl PBS and assessed with fluorescence-activated cell sorting (FACS) analysis. Data were analyzed using the FlowJo software. Gating strategy was determined based on isotype control staining.Third-passage mouse ADSCs were trypsinized, washed, and resuspended with PBS and incubated with antibody CD45 , CD44 , CD105 , or CD34  conjugated with fluorescent dyes or isotype-matched IgG controls. Single-cell suspensions were incubated at 4°C for 30 min and then washed three times with PBS. After centrifugation, the cells were resuspended in 500 4) were seeded in a 10 cm dish and cultured for up to 14 days. Then, cells were washed with PBS, fixed with 4% paraformaldehyde in PBS, and stained with 0.5% crystal violet  in methanol for 10 min at room temperature.A 3--2,5-diphenyl-tetrazolium bromide (MTT) assay was performed to evaluate cell proliferation rate at different time points. For the colony formation assay, ADSCs , 10 μm insulin , 200 μm indomethacin , and 0.5 mm 3-isobutyl-1-methylxanthine . Oil red O staining as an indicator of intracellular lipid accumulation was performed, as described previously [β-glycerophosphate , and 50 μM ascorbic acid . To quantify ALP enzymatic activity, cells were cultured for 7 days, then washed three times with PBS, fixed with 4% paraformaldehyde for 2 minutes, and visualized by staining with an alkaline phosphatase detection kit .ADSCs were cultured in complete culture media in 6-well culture plates. Adipogenic differentiation was induced for 7 days in DMEM/F12  supplemented with 10% fetal bovine serum , 1 eviously . For quaγ , rabbit anti-Notch3 , rabbit anti-c/EBPα , and mouse anti-β-actin . The membranes were incubated with HRP-conjugated secondary anti-mouse or anti-rabbit antibodies and visualized via an enhanced chemiluminescence (ECL) kit  using ChemiDoc XRS with Quantity One Software . Bands on immunoblots were analyzed by densitometry.The cells were washed twice with ice-cold PBS and lysed in RIPA buffer. The protein content of the lysate was determined using a BCA protein assay kit . The proteins were separated by 10% SDS polyacrylamide gels and transferred onto polyvinyl difluoride membranes. The membranes were blocked in 5% BSA in TBST for 1 hour at room temperature and then incubated with a primary antibody overnight at 4°C. The primary antibodies used were mouse anti-Oct-4 , rabbit anti-Sox-2 , rabbit anti-Nanog , anti-PPAR-The synthetic miR-150 mimics and negative controls were purchased from Invitrogen. Subconfluent ADSCs were washed with PBS and incubated in trypsin/EDTA, PBS, and collagenase. The cells were resuspended and pelleted at 800 g for 5 min and then resuspended in Opti-MEM® I Reduced Serum Medium . ADSCs cultured in six-well plates were transfected with miRNA mimics and siRNA using siPORT™ NeoFX™ Transfection Agent  according to the manufacturer's protocol.A small hairpin (sh) RNA Notch3 expression vector  or negative control was generated by company . The packaged lentiviruses used contained lentiviruses targeting Notch3 (shNotch3-1 and shNotch3-2) and the scrambled control (shNC). The shRNA target sequences were as follows: 5′-GCACCCAGTCCGCCCTGAGCAAATTCAAGAGATTTGCTCAGGGCGGACTGGGTGCTTTTT-3′ (shNC), 5′-GATCCGCGTGTGTAGACGGTGTCAATATTCAAGAGATATTGACACCGTCTACACACGTTTTTTA-3′ (Notch3-siR1), and 5′-GATCCGCTAGCTTCTCGTGTGCTTGTTTCAAGAGAACAAGCACACGAGAAGCTAGCTTTTTTA-3′ (Notch3-siR2).2 for 48 h and harvested for further studies. Under these circumstances, the transduction efficiency of the adenovirus reached almost 100%.For the overexpression of miR-150, recombinant adenovirus expressing miR-150 (adv-miR-150) and empty control adenovirus were constructed by Shandong Vigene Biosciences. The infection of adenovirus was performed in serum-free media. After 4 h, medium change was carried out with serum-containing fresh media. Cells were maintained at 37°C in a humidified atmosphere with 5% COThe synthetic fragment of Notch3 3′UTR containing the predicted seed match site with miR-150 or the mutant site was inserted between the SacI and XhoI cleavage sites of pmirGLO plasmid , downstream of the renilla luciferase reporter gene. For luciferase report assay, the ADSCs were cotransfected with luciferase reporters, either wild-type or mutant Notch3 3′UTR, in combination with miR-150 mimics or negative control by using Lipofectamine 2000 . After 48 hours, the firefly and renilla luciferase activities were measured and analyzed according to the manufacturer's instruction . Renilla luciferase activity was normalized to firefly luciferase activity.Total RNA was extracted from ADSCs using Trizol  according to the manufacturer's instructions. cDNA was synthesized using the RiboTM Reverse Transcription Kit . The reverse transcription reaction for miR-150 was carried out with Bulge-LoopTM miRNA qRT-PCR Primers . Relative expression was determined using U6  as an internal control for miR-150. Quantitative real-time PCR was performed with three technical replicates on the ABI-7900HT Real-Time PCR System (Applied Biosystems) using the SYBR Green Master Mix .t-test was used for comparisons between two groups. P values < 0.05 were considered to be statistically significant.All the data were analyzed by one-way analysis of variance (ANOVA). The unpaired ADSCs were isolated from the inguinal adipose tissue of C57 mice, cultured, and identified by flow cytometry using CD markers. The expression levels of blood cell-related marker CD45 and the hematopoietic stem cell marker CD34 were negative, while the mesenchymal stem cell-related markers CD44 and CD105 were highly expressed in isolated cells  showed that adipogenic differentiation of ADSCs was enhanced as a result of Notch3 inhibition  were infected with or without addition of adv-miR-150, then induced by adipogenic differentiation medium for 7 days. Western blot result showed that overexpression of miR-150 downregulated the expression of Notch3 in ADSCs. In addition, miR-150 overexpression significantly increased lipid accumulation in hADSCs and the expression of PPAR-in ADSCs . The resin ADSCs .To investigate whether miR-150 regulates self-renewal in ADSCs, the primary WT and miR-150 KO ADSCs were cultured, and we performed the MTT assay and the clonogenic assay. The results showed that cell proliferation was increased in ADSCs of miR-150 KO mice compared with WT mice. At the same time, there were more colonies in ADSCs of miR-150 KO compared with WT mice Figures .However, with miR-150 overexpression, the cell proliferation of ADSCs was decreased. In addition, the expression of the pluripotency-associated transcription factor Nanog was significantly decreased in ADSCs with miR-150 overexpression, while Sox-2 and Oct-4 levels were unchanged .This finding indicated that miR-150 suppressed ADSCs' proliferation potential.α and PPAR-γ expression and lipid formation in ADSCs with adipogenic induction, indicating increased adipogenic differentiation. Furthermore, overexpression of miR-150 significantly decreased Notch3 expression in ADSCs. Moreover, miR-150 could affect ADSCs' proliferation. This study provided the first evidence that miR-150 regulated ADSCs' proliferation and differentiation.miRNAs are a class of evolutionarily conserved endogenous noncoding single-stranded small RNA molecules. miRNAs are widely present in mammals and have a length of about 22 nt. The corresponding protein-coding genes are posttranscriptionally regulated by incomplete alignment with the 3′ untranslated region (3′UTR) sequence of the target mRNA. The most representative role of miRNAs is to induce specific targeted mRNA degradation or repression –8. In soα (PGC-1α) [Recent studies highlight that miR-150 is closely associated with lipid metabolism. In adipose tissue, miR-150 may regulate lipid metabolism and cytokine expression by targeting genes linked to lipid accumulation, such as adiponectin receptor 2 (AdipoR2) and peroxisome proliferator-activated receptor gamma coactivator-1(PGC-1α) , 20. In (PGC-1α) . miR-150(PGC-1α) . Howeverγ and inhibits the accumulation of intracellular lipid [γ-secretase inhibitor of Notch signaling could increase the adipogenesis of mouse ADSCs by promotion of PPAR-γ expression [γ.To determine how miR-150 acts as an adipogenic-promoting gene, we searched for physiological targets using bioinformatic analysis and found a 7 nt match site complementary to miR-150 in the 3′UTR of Notch3 mRNA. In the current study, dual luciferase reporter assay showed that miR-150 directly targeted Notch3 at the translational level. We found that overexpression of miR-150 and transduction of miR-150 inhibitors have opposing effects on Notch3 expression. The Notch signaling pathway consists of four transmembrane Notch receptors (Notch1-Noth4) and ligands  . Osathanar lipid . Data frar lipid . Previoupression . These fMorphologically, ADSCs from WT mice displayed as larger, with an irregular and flat shape. However, ADSCs from miR-150 KO mice displayed as spindle shape and flat shape together. MTT and colony formation experiments showed that miR-150 KO could increase the cell proliferation. To further understand the mechanism, with miR-150 overexpression in ADSCs, the level of transcription factors Oct-4, Sox-2, and Nanog was investigated, which played a key role in stem cell self-renewal and pluripotency states . Our resIn summary, this study provides the first evidence that miR-150 is expressed in ADSCs. The novel relationship between miR-150 and Notch3 is critical for adipogenesis. A new role for miR-150 in the promotion of adipogenic differentiation in ADSCs is mediated by inhibiting Notch3 signaling.