Discovery of cytosine deaminases enables base-resolution methylome mapping using a single enzyme.

Deaminases have important uses in modification detection and genome editing. However, the range of applications is limited by the small number of characterized enzymes. To expand the toolkit of deaminases, we developed an in vitro approach that bypasses a major hurdle with their toxicity in cells. We assayed 175 putative cytosine deaminases on a variety of substrates and found a broad range of activity on double- and single-stranded DNA in various sequence contexts, including CpG-specific deaminases and enzymes without sequence preference. We also characterized enzyme selectivity across six DNA modifications and reported enzymes that do not deaminate modified cytosines. The detailed analysis of diverse deaminases opens new avenues for biotechnological and medical applications. As a demonstration, we developed SEM-seq, a non-destructive single-enzyme methylation sequencing method using a modification-sensitive double-stranded DNA deaminase. The streamlined protocol enables accurate, base-resolution methylome mapping of scarce biological material, including cell-free DNA and 10 pg input DNA.

USP5 promotes lipopolysaccharide-induced apoptosis and inflammatory response by stabilizing the TXNIP protein.

BACKGROUND: The role of thioredoxin-interacting protein (TXNIP) in lipopolysaccharide-induced liver injury in mice has been reported, but the underlying mechanisms are poorly understood. METHODS: We overexpressed deubiquitinase in cells overexpressing TXNIP and then detected the level of TXNIP to screen out the deubiquitinase regulating TXNIP; the interaction between TXNIP and deubiquitinase was verified by coimmunoprecipitation. After knockdown of a deubiquitinase and overexpression of TXNIP in Huh7 and HepG2 cells, lipopolysaccharide was used to establish a cellular inflammatory model to explore the role of deubiquitinase and TXNIP in hepatocyte inflammation. RESULTS: In this study, we discovered that ubiquitin-specific protease 5 (USP5) interacts with TXNIP and stabilizes it through deubiquitylation in Huh-7 and HepG2 cells after treatment with lipopolysaccharide. In lipopolysaccharide-treated Huh-7 and HepG2 cells, USP5 knockdown increased cell viability, reduced apoptosis, and decreased the expression of inflammatory factors, including NLRP3, IL-1ß, IL-18, ASC, and procaspase-1. Overexpression of TXNIP reversed the phenotype induced by knockdown USP5. CONCLUSIONS: In summary, USP5 promotes lipopolysaccharide-induced apoptosis and inflammatory response by stabilizing the TXNIP protein.

Investigating the effect of Icaritin on hepatocellular carcinoma based on network pharmacology.

Hepatocellular carcinoma is one of the cancers that kill people in the global population. Icaritin, a small molecule drug approved by NMPA, has demonstrated potential anti-HCC effects. However, its underlying molecular mechanisms remain unclear. We employed a multi-omics approach in this study, including pharmaco-omics and proteomics, to look into the Icaritin's possible molecular targets and workings in the therapy of HCC. Through pharmaco-omics analysis, we identified ten putative target genes of Icaritin, including FYN. The relationship between Icaritin and these target genes, particularly FYN, was further validated through in vitro and in vivo experiments. The outcomes revealed that Icaritin may exert its anti-HCC effects through modulating the FYN gene, highlighting the importance of multi-omics approaches in drug discovery research. This research gives valuable insights regarding the therapeutic potential of Icaritin against HCC and its possible molecular mechanisms.

SNRPD1 inhibition suppresses the proliferation of hepatocellular carcinoma and promotes autophagy through the PI3K/AKT/mTOR/4EBP1 pathway.

BACKGROUND: Small nuclear ribonucleoprotein Sm D1 (SNRPD1) has been reported as an oncogene in some solid cancers. Our previous study suggested that SNRPD1 has diagnostic and prognostic value in hepatocellular carcinoma (HCC), but its role in tumor growth and biological behavior remains unknown. In this study, we aimed to unravel the role and mechanism of SNRPD1 in HCC. METHODS: We investigated the SNRPD1 mRNA level in adjacent normal liver tissues and HCC tissues with different tumor stages in the UALCAN database. The associations between SNRPD1 mRNA expression and HCC prognosis were investigated in TCGA database. Then, 52 pairs of frozen HCC tissues and corresponding adjacent normal liver tissues were collected to perform qPCR and immunohistochemistry assay. Next, we carried out a series of experiments in vitro and in vivo to investigate the effects of SNRPD1 expression on cell invasion, migration, proliferation, autophagy, and the PI3K/AKT/mTOR pathway. RESULTS: The bioinformatics analysis and qPCR in our patient cohort demonstrated that the SNRPD1 mRNA level in HCC tissues was higher than in adjacent normal tissues. In addition, the immunohistochemistry assay exhibited an increased SNRPD1 protein level with the tumor stage increase. Survival analysis suggested that higher expression of SNRPD1 was significantly associated with unfavorable prognosis of patients with HCC. The functional experiments in vitro indicated that SNRPD1 knockdown suppressed the cellular proliferation, migration, and invasion capacities. Furthermore, SNRPD1 inhibition induced cellular apoptosis and arrested the HCC cells at the G0/G1 phase of the cell cycle. Mechanistic analyses demonstrated that SNRPD1 knockdown induced the increase of autophagic vacuoles and the expression of autophagy-related genes (ATG5, ATG7, and ATG12) and blocked the PI3K/AKT/mTOR/4EBP1 pathway in vitro. Moreover, SNRPD1 inhibition suppressed tumor growth and expression of the Ki67 protein in vivo. CONCLUSIONS: SNRPD1 may serve as an oncogene in HCC and promote tumor proliferation via inhibiting autophagy induced through the PI3K/Akt/mTOR/4EBP1 pathway.

Defining the filarial N-glycoproteome by glycosite mapping in the human parasitic nematode Brugia malayi.

N-linked glycosylation is a critical post translational modification of eukaryotic proteins. N-linked glycans are present on surface and secreted filarial proteins that play a role in host parasite interactions. Examples of glycosylated Brugia malayi proteins have been previously identified but there has not been a systematic study of the N-linked glycoproteome of this or any other filarial parasite. In this study, we applied an enhanced N-glyco FASP protocol using an engineered carbohydrate-binding protein, Fbs1, to enrich N-glycosylated peptides for analysis by LC-MS/MS. We then mapped the N-glycosites on proteins from three host stages of the parasite: adult female, adult male and microfilariae. Fbs1 enrichment of N-glycosylated peptides enhanced the identification of N-glycosites. Our data identified 582 N-linked glycoproteins with 1273 N-glycosites. Gene ontology and cell localization prediction of the identified N-glycoproteins indicated that they were mostly membrane and extracellular proteins. Comparing results from adult female worms, adult male worms, and microfilariae, we find variability in N-glycosylation at the protein level as well as at the individual N-glycosite level. These variations are highlighted in cuticle N-glycoproteins and adult worm restricted N-glycoproteins as examples of proteins at the host parasite interface that are well positioned as potential therapeutic targets or biomarkers.

Structures, activity and mechanism of the Type IIS restriction endonuclease PaqCI.

Type IIS restriction endonucleases contain separate DNA recognition and catalytic domains and cleave their substrates at well-defined distances outside their target sequences. They are employed in biotechnology for a variety of purposes, including the creation of gene-targeting zinc finger and TAL effector nucleases and DNA synthesis applications such as Golden Gate assembly. The most thoroughly studied Type IIS enzyme, FokI, has been shown to require multimerization and engagement with multiple DNA targets for optimal cleavage activity; however, details of how it or similar enzymes forms a DNA-bound reaction complex have not been described at atomic resolution. Here we describe biochemical analyses of DNA cleavage by the Type IIS PaqCI restriction endonuclease and a series of molecular structures in the presence and absence of multiple bound DNA targets. The enzyme displays a similar tetrameric organization of target recognition domains in the absence or presence of bound substrate, with a significant repositioning of endonuclease domains in a trapped DNA-bound complex that is poised to deliver the first of a series of double-strand breaks. PaqCI and FokI share similar structural mechanisms of DNA cleavage, but considerable differences in their domain organization and quaternary architecture, facilitating comparisons between distinct Type IIS enzymes.

Comparative site-specific N-glycoproteome analysis reveals aberrant N-glycosylation and gives insights into mannose-6-phosphate pathway in cancer.

N-glycosylation is implicated in cancers and aberrant N-glycosylation is recognized as a hallmark of cancer. Here, we mapped and compared the site-specific N-glycoproteomes of colon cancer HCT116 cells and isogenic non-tumorigenic DNMT1/3b double knockout (DKO1) cells using Fbs1-GYR N-glycopeptide enrichment technology and trapped ion mobility spectrometry. Many significant changes in site-specific N-glycosylation were revealed, providing a molecular basis for further elucidation of the role of N-glycosylation in protein function. HCT116 cells display hypersialylation especially in cell surface membrane proteins. Both HCT116 and DKO1 show an abundance of paucimannose and 80% of paucimannose-rich proteins are annotated to reside in exosomes. The most striking N-glycosylation alteration was the degree of mannose-6-phosphate (M6P) modification. N-glycoproteomic analyses revealed that HCT116 displays hyper-M6P modification, which was orthogonally validated by M6P immunodetection. Significant observed differences in N-glycosylation patterns of the major M6P receptor, CI-MPR in HCT116 and DKO1 may contribute to the hyper-M6P phenotype of HCT116 cells. This comparative site-specific N-glycoproteome analysis provides a pool of potential N-glycosylation-related cancer biomarkers, but also gives insights into the M6P pathway in cancer.

miR-6071 inhibits hepatocellular carcinoma progression via targeting PTPN11.

Hepatocellular carcinoma (HCC) is a deadly malignancy. Liver cancer stem cells (LCSCs) participated in HCC progression and caused failure of chemotherapy. However, the underlying mechanism for the LCSCs regulation was unclear. In this study, we found that miR-6071 expression was decreased in LCSCs. Gain-of-function assays showed that miR-6071 overexpression repressed LCSCs self-renewal and tumorigenesis and inhibited HCC cells proliferation and migration. In mechanism, bioinformatics and luciferase reporter assay demonstrated that miR-6071 targeted 3'UTR of PTPN11 mRNA. Pearson analysis revealed a negative correlation between miR-6071 expression and PTPN11 levels in HCC tissue samples. Further study showed that PTPN11 interference and specific inhibitors IACS-13909 abrogated the discrepancy of self-renewal ability, proliferation, migration and tumorigenicity capacity between miR-6071 overexpression HCC cells and control cells. Moreover, upregulation of miR-6071 sensitized HCC cells to lenvatinib treatment. Clinical cohort analysis revealed that HCC patients with high miR-6071 expression got more survival benefit from postoperative lenvatinib treatment than patients with low miR-6071 levels. In conclusion, our study demonstrated a regulation mechanism of LCSCs, a target against LSCSs, and a biomarker for postoperative lenvatinib treatment.

Retracted: Expression of Long Non-Coding RNA (lncRNA) Small Nucleolar RNA Host Gene 1 (SNHG1) Exacerbates Hepatocellular Carcinoma Through Suppressing miR-195.

Retracted, due to breach of publishing guidelines, following the identification of non-original and manipulated figure images. Reference: Hui Zhang, Dong Zhou, Mingang Ying, Minyong Chen, Peng Chen, Zhaoshuo Chen, Fan Zhang: Expression of Long Non-Coding RNA (lncRNA) Small Nucleolar RNA Host Gene 1 (SNHG1) Exacerbates Hepatocellular Carcinoma Through Suppressing miR-195. Med Sci Monit, 2016; 22: 4820-4829. DOI: 10.12659/MSM.898574.

Small Nucleolar RNA Host Gene 1 (SNHG1) and Chromosome 2 Open Reading Frame 48 (C2orf48) as Potential Prognostic Signatures for Liver Cancer by Constructing Regulatory Networks.

BACKGROUND Liver cancer is a common malignant tumor with poor prognosis. The present study sought to identify potential signatures that can predict the prognosis of patients with liver cancer. MATERIAL AND METHODS The RNA sequencing (RNA-seq) and clinical information of liver cancer patients were obtained from the Cancer Genome Atlas (TCGA) database. Differentially expressed long noncoding RNAs (lncRNAs), microRNAs (miRNAs), and messenger RNAs (mRNAs) were identified between liver cancer and adjacent normal tissues. After predicting lncRNA-miRNA and miRNA-mRNA pairs using online databases, the competing endogenous RNA (ceRNA) networks were constructed. Furthermore, the prognostic value of these differentially expressed genes was evaluated using univariate and multivariate Cox regression analyses. RESULTS After constructing the ceRNA network, 2 lncRNAs small nucleolar RNA host gene 1 (SNHG1) and chromosome 2 open reading frame 48 (C2orf48) with the most nodes were identified. Correlation analysis revealed that SNHG1 was correlated with miR-195 and C2orf48 was correlated with miR-195 and miR-93. High expression of SNHG1, C2orf48, and miR-93 can contribute to poorer clinical outcomes compared to low expression. Furthermore, low miR-195 expression was correlated with shorter survival time than was high expression. SNHG1 and C2orf48 were closely associated with histology grade. Univariate and multivariate Cox regression analyses confirmed that SNHG1 and C2orf48 are risk factors for liver cancer. CONCLUSIONS Our findings revealed that SNHG1 and C2orf48 possess potential prognostic value and should be considered as possible biomarkers for predicting clinical outcomes for patients with liver cancer.

Association between the expression of carbonic anhydrase II and clinicopathological features of hepatocellular carcinoma.

The present study aimed to examine the molecular marker associated with the therapy and prognosis of hepatocellular carcinoma (HCC), and further investigate the association between its expression and the clinicopathological features of HCC. To select the core genes closely associated with HCC, differentially expressed genes (DEGs) were analyzed and screened from Gene Expression Omnibus datasets (GSE 36376) using a bioinformatics approach. Tumor and adjacent tissues were collected form 112 patients of HCC who were treated by radical resection. The expression levels of carbonic anhydrase II (CA2) in the tumor and adjacent tissues were determined using reverse transcription-quantitative polymerase chain reaction analysis and immunohistochemistry. The χ2 test was applied for observing the association between the expression of CA2 and clinicopathological features of patients with HCC. The effects of the expression of CA2 on the patients' overall survival (OS) and disease-free survival (DFS) were examined via Kaplan-Meier analysis. A total of 83 DEGs were screened and analyzed using gene network analysis, among which CA2 had direct interactions with more than one disease gene of HCC. The results of immunohistochemistry showed that CA2 was expressed at a lower level in the tumor tissues compared with the adjacent tissues (t=3.012, P=0.010). Single factor analysis revealed that the mRNA expression of CA2 was able to predict the recurrence of HCC, and was significantly associated with α-fetoprotein (AFP), microvascular invasion, tumor-node-metastasis (TNM) staging, and recurrence (P<0.05). The expression levels of AFP, CA2 and TNM staging were confirmed to be independent prognostic factors of HCC (P<0.05). Kaplan-Meier analysis demonstrated that the group with a high expression of CA2 showed increased DFS and OS, compared with the low expression group (P<0.05). These findings indicated that elevated CA2 increased DFS and OS of HCC, which suggested that CA2 may be a potential target for HCC therapy.

Relationship between drug resistance andABCB1 gene in pancreatic cancer / 中国肿瘤生物治疗杂志

@#Objective: To investigate the relationship between drug resistance and expression of ABC-binding cassette subfamily B member 1 (ABCB1) as well as its promoter methylation in pancreatic cancer. Methods: Fifteen pairs of pancreatic cancerous tissues and corresponding para-cancerous tissues, which were pathologically verified in Fujian Cancer Hospital from August 2015 to August 2018, were collected for this study; in addition, 3 cases of normal pancreatic tissues and pancreatic cancer cell line SW1990 were also collected. Gemcitabine (GEM)-resistant human pancreatic cancer cell line SW1990/GZ was induced by intermittent concentration gradient multiplication method. The expression level ofABCB1 in SW1990 cells, SW1990/GZ cells, pancreatic cancer tissues and apara-cancerous tissues was detected by qPCR. Methylation of ABCB1 promoter region in SW1990 cells, SW1990/GZ cells and pancreatic cancerous tissues was determined by MSP-PCR. Results: Compared with SW1990 cells, the morphology of SW1990/GZ cells showed more vacuoles, more mitotic images, clumpy growth and increased drug resistance (P<0.05). ABCB1 expression in pancreatic cancer tissues was significantly higher than that in para-cancerous tissues (P<0.01). The expression of ABCB1 in SW1990 and SW1990/GZ cells was significantly higher than that in normal pancreatic tissues (P<0.05 or P<0.01), and the expression of ABCB1 in SW1990/GZ cells was higher than that in SW1990 cells (P<0.05). ABCB1 promoters in SW1990, SW1990/GZ cells and normal pancreatic tissues were hypomethylated. Rate of methylation in pancreatic can cerous tissues and normalpancreatic tissues was 6.7%(1/15) and 0.00%(0/3) respectively,and the difference was statistically insignificant (all P>0.05). Conclusion: Increased ABCB1 expression in pancreatic cancer tissues and cells is associated with drug resistance, but its gene expression does not depend on promoter methylation regulation.

ß-arrestin 1 regulates ß2-adrenergic receptor-mediated skeletal muscle hypertrophy and contractility.

BACKGROUND: ß2-adrenergic receptors (ß2ARs) are the target of catecholamines and play fundamental roles in cardiovascular, pulmonary, and skeletal muscle physiology. An important action of ß2AR stimulation on skeletal muscle is anabolic growth, which has led to the use of agonists such as clenbuterol by athletes to enhance muscle performance. While previous work has demonstrated that ß2ARs can engage distinct signaling and functional cascades mediated by either G proteins or the multifunctional adaptor protein, ß-arrestin, the precise role of ß-arrestin in skeletal muscle physiology is not known. Here, we tested the hypothesis that agonist activation of the ß2AR by clenbuterol would engage ß-arrestin as a key transducer of anabolic skeletal muscle growth. METHODS: The contractile force of isolated extensor digitorum longus muscle (EDL) and calcium signaling in isolated flexor digitorum brevis (FDB) fibers were examined from the wild-type (WT) and ß-arrestin 1 knockout mice (ßarr1KO) followed by chronic administration of clenbuterol (1 mg/kg/d). Hypertrophic responses including fiber composition and fiber size were examined by immunohistochemical imaging. We performed a targeted phosphoproteomic analysis on clenbuterol stimulated primary cultured myoblasts from WT and ßarr1KO mice. Statistical significance was determined by using a two-way analysis with Sidak's or Tukey's multiple comparison test and the Student's t test. RESULTS: Chronic administration of clenbuterol to WT mice enhanced the contractile force of EDL muscle and calcium signaling in isolated FDB fibers. In contrast, when administered to ßarr1KO mice, the effect of clenbuterol on contractile force and calcium influx was blunted. While clenbuterol-induced hypertrophic responses were observed in WT mice, this response was abrogated in mice lacking ß-arrestin 1. In primary cultured myoblasts, clenbuterol-stimulated phosphorylation of multiple pro-hypertrophy proteins required the presence of ß-arrestin 1. CONCLUSIONS: We have identified a previously unappreciated role for ß-arrestin 1 in mediating ß2AR-stimulated skeletal muscle growth and strength. We propose these findings could have important implications in the design of future pharmacologic agents aimed at reversing pathological conditions associated with skeletal muscle wasting.

ß-Arrestin2 mediates progression of murine primary myelofibrosis.

Primary myelofibrosis is a myeloproliferative neoplasm associated with significant morbidity and mortality, for which effective therapies are lacking. ß-Arrestins are multifunctional adaptor proteins involved in developmental signaling pathways. One isoform, ß-arrestin2 (ßarr2), has been implicated in initiation and progression of chronic myeloid leukemia, another myeloproliferative neoplasm closely related to primary myelofibrosis. Accordingly, we investigated the relationship between ßarr2 and primary myelofibrosis. In a murine model of MPLW515L-mutant primary myelofibrosis, mice transplanted with donor ßarr2-knockout (ßarr2-/-) hematopoietic stem cells infected with MPL-mutant retrovirus did not develop myelofibrosis, whereas controls uniformly succumbed to disease. Although transplanted ßarr2-/- cells homed properly to marrow, they did not repopulate long-term due to increased apoptosis and decreased self-renewal of ßarr2-/- cells. In order to assess the effect of acute loss of ßarr2 in established primary myelofibrosis in vivo, we utilized a tamoxifen-induced Cre-conditional ßarr2-knockout mouse. Mice that received Cre (+) donor cells and developed myelofibrosis had significantly improved survival compared with controls. These data indicate that lack of antiapoptotic ßarr2 mediates marrow failure of murine hematopoietic stem cells overexpressing MPLW515L. They also indicate that ßarr2 is necessary for progression of primary myelofibrosis, suggesting that it may serve as a novel therapeutic target in this disease.

Hepatic ß-arrestin 2 is essential for maintaining euglycemia.

An increase in hepatic glucose production (HGP) represents a key feature of type 2 diabetes. This deficiency in metabolic control of glucose production critically depends on enhanced signaling through hepatic glucagon receptors (GCGRs). Here, we have demonstrated that selective inactivation of the GPCR-associated protein ß-arrestin 2 in hepatocytes of adult mice results in greatly increased hepatic GCGR signaling, leading to striking deficits in glucose homeostasis. However, hepatocyte-specific ß-arrestin 2 deficiency did not affect hepatic insulin sensitivity or ß-adrenergic signaling. Adult mice lacking ß-arrestin 1 selectively in hepatocytes did not show any changes in glucose homeostasis. Importantly, hepatocyte-specific overexpression of ß-arrestin 2 greatly reduced hepatic GCGR signaling and protected mice against the metabolic deficits caused by the consumption of a high-fat diet. Our data support the concept that strategies aimed at enhancing hepatic ß-arrestin 2 activity could prove useful for suppressing HGP for therapeutic purposes.

An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides.

A method for selective and comprehensive enrichment of N-linked glycopeptides was developed to facilitate detection of micro-heterogeneity of N-glycosylation. The method takes advantage of the inherent properties of Fbs1, which functions within the ubiquitin-mediated degradation system to recognize the common core pentasaccharide motif (Man3GlcNAc2) of N-linked glycoproteins. We show that Fbs1 is able to bind diverse types of N-linked glycomolecules; however, wild-type Fbs1 preferentially binds high-mannose-containing glycans. We identified Fbs1 variants through mutagenesis and plasmid display selection, which possess higher affinity and improved recovery of complex N-glycomolecules. In particular, we demonstrate that the Fbs1 GYR variant may be employed for substantially unbiased enrichment of N-linked glycopeptides from human serum. Most importantly, this highly efficient N-glycopeptide enrichment method enables the simultaneous determination of N-glycan composition and N-glycosites with a deeper coverage (compared to lectin enrichment) and improves large-scale N-glycoproteomics studies due to greatly reduced sample complexity.

Expression of Long Non-Coding RNA (lncRNA) Small Nucleolar RNA Host Gene 1 (SNHG1) Exacerbates Hepatocellular Carcinoma Through Suppressing miR-195.

BACKGROUND Aberrant expression of lncRNA has been suggested to have an association with tumorigenesis. Our study was designed to reveal the underlying connection between lncRNA SNHG1 and hepatocellular carcinoma (HCC) pathogenesis. MATERIAL AND METHODS A total of 122 pairs of HCC tissues (case group) and matched adjacent non-tumor liver tissues (control group) were collected for this study. RT-PCR and in situ hybridization were conducted to investigate differences in lncRNA SNHG1 expression between the case and control group. The expression levels of lncRNA SNHG1 and miR-195 in HepG2 cells transfected with SNHG1-mimic and SNHG1-inhibitor were measured by RT-PCR. The proliferation, invasion, and migration status of HepG2 cells after transfection were assessed through MTT assay, wound healing assay, and Transwell assay, respectively. Whether miR-195 is a direct downstream target of lncRNA SNHG1 was verified by both bioinformatics target gene prediction and dual-luciferase report assay. RESULTS The expression level of lncRNA SNHG1 was remarkably upregulated in HCC tissues and cell lines compared with normal tissues and cell lines. High expression of lncRNA SNHG1 contributed to the downregulation of miR-195 in HepG2 cells. Also, lncRNA SNHG1 exacerbated HCC cell proliferation, invasion, and migration in vitro through the inhibition of miR-195. This suggests that miR-195 is a direct downstream target of lncRNA SNHG1. CONCLUSIONS lncRNA SNHG1 may contribute to the aggravation of HCC through the inhibition of miR-195.

A DsbA-Deficient Periplasm Enables Functional Display of a Protein with Redox-Sensitive Folding on M13 Phage.

The requirements for target protein folding in M13 phage display are largely underappreciated. Here we chose Fbs1, a carbohydrate binding protein, as a model to address this issue. Importantly, folding of Fbs1 is impaired in an oxidative environment. Fbs1 can be displayed on M13 phage using the SRP or Sec pathway. However, the displayed Fbs1 protein is properly folded only when Fbs1 is translocated via the SRP pathway and displayed using Escherichia coli cells with a DsbA-negative periplasm. This study indicates M13 phage display may be improved using a system specifically designed according to the folding requirements of each target protein.

Structure-activity studies of Wnt/ß-catenin inhibition in the Niclosamide chemotype: Identification of derivatives with improved drug exposure.

The Wnt signaling pathway plays a key role in regulation of organ development and tissue homeostasis. Dysregulated Wnt activity is one of the major underlying mechanisms responsible for many diseases including cancer. We previously reported the FDA-approved anthelmintic drug Niclosamide inhibits Wnt/ß-catenin signaling and suppresses colon cancer cell growth in vitro and in vivo. Niclosamide is a multi-functional drug that possesses important biological activity in addition to inhibition of Wnt/ß-catenin signaling. Here, we studied the SAR of Wnt signaling inhibition in the anilide and salicylamide region of Niclosamide. We found that the 4'-nitro substituent can be effectively replaced by trifluoromethyl or chlorine and that the potency of inhibition was dependent on the substitution pattern in the anilide ring. Non-anilide, N-methyl amides and reverse amide derivatives lost significant potency, while acylated salicylamide derivatives inhibited signaling with potency similar to non-acyl derivatives. Niclosamide's low systemic exposure when dosed orally may hinder its use to treat systemic disease. To overcome this limitation we identified an acyl derivative of Niclosamide, DK-520 (compound 32), that significantly increased both the plasma concentration and the duration of exposure of Niclosamide when dosed orally. The studies herein provide a medicinal chemical foundation to improve the pharmacokinetic exposure of Niclosamide and Wnt-signaling inhibitors based on the Niclosamide chemotype. The identification of novel derivatives of Niclosamide that metabolize to Niclosamide and increase its drug exposure may provide important research tools for in vivo studies and provide drug candidates for treating cancers with dysregulated Wnt signaling including drug-resistant cancers. Moreover, since Niclosamide is a multi-functional drug, new research tools such as DK520 could directly result in novel treatments against bacterial and viral infection, lupus, and metabolic diseases such as type II diabetes, NASH and NAFLD.

Circulating Exosomes Induced by Cardiac Pressure Overload Contain Functional Angiotensin II Type 1 Receptors.

BACKGROUND: Whether biomechanical force on the heart can induce exosome secretion to modulate cardiovascular function is not known. We investigated the secretion and activity of exosomes containing a key receptor in cardiovascular function, the angiotensin II type I receptor (AT1R). METHODS AND RESULTS: Exosomes containing AT1Rs were isolated from the media overlying AT1R-overexpressing cells exposed to osmotic stretch and from sera of mice undergoing cardiac pressure overload. The presence of AT1Rs in exosomes was confirmed by both electron microscopy and radioligand receptor binding assays and shown to require ß-arrestin2, a multifunctional adaptor protein essential for receptor trafficking. We show that functional AT1Rs are transferred via exosomes in an in vitro model of cellular stretch. Using mice with global and cardiomyocyte conditional deletion of ß-arrestin2, we show that under conditions of in vivo pressure overload the cellular source of the exocytosis of exosomes containing AT1R is the cardiomyocyte. Exogenously administered AT1R-enriched exosomes target cardiomyocytes, skeletal myocytes, and mesenteric resistance vessels and are sufficient to confer blood pressure responsiveness to angiotensin II infusion in AT1R knockout mice. CONCLUSIONS: AT1R-enriched exosomes are released from the heart under conditions of in vivo cellular stress to likely modulate vascular responses to neurohormonal stimulation. In the context of the whole organism, the concept of G protein-coupled receptor trafficking should consider circulating exosomes as part of the reservoir of functional AT1Rs.