Neuroblast Differentiation-associated Protein Research Articles

KLF7 promotes neuroblastoma differentiation through the GTPase signaling pathway by upregulating neuroblast differentiation-associated protein AHNAKs and glycerophosphodiesterase GDPD5.

The arrest of neural crest-derived sympathoadrenal neuroblast differentiation contributes to neuroblastoma formation, and overriding this blocked differentiation is a clear strategy for treating high-risk neuroblastoma. A better understanding of neuroblast or neuroblastoma differentiation is essential for developing new therapeutic approaches. It has been proposed that Krueppel-like factor 7 (KLF7) is a neuroblastoma super-enhancer-associated transcription factor gene. Moreover, KLF7 was found to be intensely active in postmitotic neuroblasts of the developing nervous system during embryogenesis. However, the role of KLF7 in the differentiation of neuroblast or neuroblastoma is unknown. Here, we find a strong association between high KLF7 expression and favorable clinical outcomes in neuroblastoma. KLF7 induces differentiation of neuroblastoma cells independently of the retinoic acid (RA) pathway and acts cooperatively with RA to induce neuroblastoma differentiation. KLF7 alters the GTPase activity and multiple differentiation-related genes by binding directly to the promoters of neuroblast differentiation-associated protein (AHNAK and AHNAK2) and glycerophosphodiester phosphodiesterase domain-containing protein 5 (GDPD5) and regulating their expression. Furthermore, we also observe that silencing KLF7 in neuroblastoma cells promotes the adrenergic-to-mesenchymal transition accompanied by changes in enhancer-mediated gene expression. Our results reveal that KLF7 is an inducer of neuroblast or neuroblastoma differentiation with prognostic significance and potential therapeutic value.

Ahnak is required to balance calcium ion homeostasis and smooth muscle development in the urinary system

BackgroundVarious renal abnormalities, including hydronephrosis, polycystic kidney disease, and hydroureter, have been reported, and these abnormalities are present in DiGeorge syndrome, renal dysplasia, and acute kidney failure. Previous studies have shown that various genes are associated with renal abnormalities. However, the major target genes of nonobstructive hydronephrosis have not yet been elucidated.ResultsWe examined neuroblast differentiation-associated protein Ahnak localization and analyzed morphogenesis in developing kidney and ureter. To investigated function of Ahnak, RNA-sequencing and calcium imaging were performed in wild type and Ahnak knockout (KO) mice. Ahnak localization was confirmed in the developing mouse kidneys and ureter. An imbalance of calcium homeostasis and hydronephrosis, which involves an expanded renal pelvis and hydroureter, was observed in Ahnak KO mice. Gene Ontology enrichment analysis on RNA-seq results indicated that ‘Channel Activity’, ‘Passive Transmembrane Transporter Activity’ and ‘Cellular Calcium Ion Homeostasis’ were downregulated in Ahnak KO kidney. ‘Muscle Tissue Development’, ‘Muscle Contraction’, and ‘Cellular Calcium Ion Homeostasis’ were downregulated in Ahnak KO ureter. Moreover, peristaltic movement of smooth muscle in the ureter was reduced in Ahnak KO mice.ConclusionsAbnormal calcium homeostasis causes renal disease and is regulated by calcium channels. In this study, we focused on Ahnak, which regulates calcium homeostasis in several organs. Our results indicate that Ahnak plays a pivotal role in kidney and ureter development, and in maintaining the function of the urinary system.

Neuroblast Differentiation-Associated Protein Derived Polypeptides: AHNAK(5758-5775) Induces Inflammation by Activating Mast Cells via ST2

ABSTRACT Psoriasis is a chronic inflammatory skin disease. Mast cells are significantly increased and activated in psoriatic lesions and are involved in psoriatic inflammation. Some endogenous substances can interact with the surface receptors of mast cells and initiate the release of downstream cytokines that participate in inflammatory reactions. Neuroblast differentiation-associated protein (AHNAK) is mainly expressed in the skin, esophagus, kidney, and other organs and participates in various biological processes in the human body. AHNAK and its derived peptides have been reported to be involved in the activation of mast cells and other immune processes. This study aimed to investigate whether AHNAK (5758–5775), a neuroblast differentiation-associated protein-derived polypeptide, could be considered a new endogenous substance in psoriasis patients, which activates mast cells and induces the skin inflammatory response contributing to psoriasis. Wild-type mice were treated with AHNAK(5758–5775) to observe the infiltration of inflammatory cells in the skin and cytokine release in vivo. The release of inflammatory mediators by mouse primary mast cells and the laboratory of allergic disease 2 (LAD2) human mast cells was measured in vitro. Molecular docking analysis, molecular dynamics simulation, and siRNA transfection were used to identify the receptor of AHNAK(5758–5775). AHNAK(5758–5775) could cause skin inflammation and cytokine release in wild-type mice and activated mast cells in vitro. Moreover, suppression of tumorigenicity 2 (ST2) might be a key receptor mediating AHNAK(5758–5775)’s effect on mast cells and cytokine release. We propose a novel polypeptide, AHNAK(5758–5775), which induces an inflammatory reaction and participates in the occurrence and development of psoriasis by activating mast cells.

AHNAK Contributes to Hepatocellular Carcinoma Growth by Interacting with IGF-1R.

Neuroblast differentiation-associated protein AHNAK, a large structural scaffold protein, remains mysterious in biological processes. AHNAK plays a suppressive or progressive role in different types of cancers. To investigate the role of the AHNAK in hepatocellular carcinoma (HCC), cell viability assays were performed to determine the cell proliferation of the stable AHNAK-knockdown HepG2 cell line; co-immunoprecipitation (Co-IP) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) were performed on HCC and matched paracancerous (MPC) tissues. The Metascape platform was used for enrichment analyses; the "ComplexHeatmap" package was applied for cluster analyses and visualization. Co-IP, Western botting and immunofluorescence double staining were performed to assess the interactions between AHNAK and insulin-like growth factor 1 receptor (IGF-1R). AHNAK silencing reduced the viability of HepG2 cells; the interactome in HCC and MPC tissues enriched 204 pathways and processes, which partially reflected the signature of HCC field cancerization. AHNAK could co-localize and interact with IGF-1R. These results suggested that the AHNAK complex contributes to HCC growth, potentially by interacting with IGF-1R.

LB873 Neuroblast differentiation-associated protein derived polypeptides: AHNAK(5758-5775) induced inflammatory reaction by activation mast cells via ST2

Mast cells significantly increase and activate in psoriasis lesions and are involved in psoriatic inflammation. Neuroblast differentiation-associated protein (AHNAK) mainly express in skin, esophagus, kidney and other organs, which participates in the differentiation of neurons, the formation of cytoskeletal structure muscular regeneration, and the calcium homeostasis process. Whether AHNAK is involved in mast cell activation is unclear, and the mechanisms of AHNAK-induced skin inflammation also need investigation.

The effect of feed supplementation with zinc glycine chelate and zinc sulphate on hepatic proteome profiles in chickens

One of the most important factors influencing production outcomes in poultry farming is optimal balancing of the feed ration in terms of macro- and micronutrients. Zinc is an essential trace element that increases metabolic processes. The natural components of poultry feed do not contain sufficient zinc, or even contain numerous factors limiting its absorption, which necessitates supplementation of feed with this element. The purpose of the study was to identify proteins synthesized in the liver of chickens fed a diet supplemented with zinc in the form of sulphates and a glycine chelate; to determine differences in the expression of these proteins; and to describe the peptide profile of hepatocytes in chickens. A total of 900 1-day-old male Ross 308 chickens, assigned to 5 dietary treatments, were used in the experiment. The experimental groups received a basal diet + zinc in inorganic and organic form and a phytase supplement. The material for analysis comprised samples of liver and serum. The zinc concentration in serum and tissues was determined by flame atomic absorption spectrometry. The liver lipid profile was estimated by the gravimetric method. A complete proteomic analysis of liver samples included protein separation by two-dimensional electrophoresis, plotting of a protein map illustrating the proteome of the liver, statistical analysis, and identification of statistically significant proteins by MALDI-TOF mass spectrometry. The results of the study demonstrate that organic and inorganic forms of zinc used as feed additives for chickens not only affect the concentration of this element in the liver, but also have a systemic effect by altering the expression of proteins. A complete proteomic analysis of liver samples made it possible to identify eight proteins whose expression depended on the type of supplement added to the feed. The results clearly indicate that bioavailable zinc administered in the form of glycine chelates affects processes stimulating synthesis of ApoA-I, β2-MG, protein-disulphide-isomerase, neuroblast differentiation-associated protein AHNAK, and alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase 1 in the tissues of chickens. Supplementation of poultry feed with zinc in both sulphate and chelated form caused changes in the expression of the protein yippee-like 4 and histone H1.1. A high concentration of prolyl endopeptidase protein in the liver of birds receiving zinc sulphate was also observed. In conclusion, supplementation of poultry feed with zinc chelates modifies the expression profiles of proteins involved in intracellular transport, the cell cycle, metabolism, and the immune response.

Multisample Mass Spectrometry-Based Approach for Discovering Injury Markers in Chronic Kidney Disease

AbstractUrinary proteomics studies have primarily focused on identifying markers of chronic kidney disease (CKD) progression. Here, we aimed to determine urinary markers of CKD renal parenchymal injury through proteomics analysis in animal kidney tissues and cells and in the urine of patients with CKD. Label-free quantitative proteomics analysis based on liquid chromatography–tandem mass spectrometry was performed on urine samples obtained from 6 normal controls and 9, 11, and 10 patients with CKD stages 1, 3, and 5, respectively, and on kidney tissue samples from a rat CKD model by 5/6 nephrectomy. Tandem mass tag-based quantitative proteomics analysis was performed for glomerular endothelial cells (GECs) and proximal tubular epithelial cells (PTECs) before and after inducing 24-h hypoxia injury. Upon hierarchical clustering, out of 858 differentially expressed proteins (DEPs) in the urine of CKD patients, the levels of 416 decreased and 403 increased sequentially according to the disease stage, respectively. Among 2965 DEPs across 5/6 nephrectomized and sham-operated rat kidney tissues, 86 DEPs showed same expression patterns in the urine and kidney tissue. After cross-validation with two external animal proteome data sets, 38 DEPs were organized; only ten DEPs, including serotransferrin, gelsolin, poly ADP-ribose polymerase 1, neuroblast differentiation-associated protein AHNAK, microtubule-associated protein 4, galectin-1, protein S, thymosin beta-4, myristoylated alanine-rich C-kinase substrate, and vimentin, were finalized by screening human GECs and PTECs data. Among these ten potential candidates for universal CKD marker, validation analyses for protein S and galectin-1 were conducted. Galectin-1 was observed to have a significant inverse correlation with renal function as well as higher expression in glomerulus with chronic injury than protein S. This constitutes the first multisample proteomics study for identifying key renal-expressed proteins associated with CKD progression. The discovered proteins represent potential markers of chronic renal cell and tissue damage and candidate contributors to CKD pathophysiology.

Phosphorylation Ratio Determination in Fresh-Frozen and Formalin-Fixed Paraffin-Embedded Tissue with Targeted Mass Spectrometry.

Formalin-fixed paraffin-embedded (FFPE) tissues are routinely prepared and collected for diagnostics in pathology departments. These are, therefore, the most accessible research sources in pathology archives. In this study we investigated whether we can apply a targeted and quantitative parallel reaction monitoring (PRM) method for FFPE tissue samples in a sensitive and reproducible way. The feasibility of this technical approach was demonstrated for normal brain and glioblastoma multiforme tissues. Two methods were used: PRM measurement of a tryptic digest without phosphopeptide enrichment (Direct-PRM) and after Fe-NTA phosphopeptide enrichment (Fe-NTA-PRM). With these two methods, the phosphorylation ratio could be determined for four selected peptide pairs that originate from neuroblast differentiation-associated protein (AHNAK S5448-p), calcium/calmodulin-dependent protein kinase type II subunit delta (CAMK2D T337-p), eukaryotic translation initiation factor 4B (EIF4B S93-p), and epidermal growth factor receptor (EGFR S1166-p). In normal brain FFPE tissues, the Fe-NTA-PRM method enabled the quantification of targeted phosphorylated peptides with high reproducibility (CV < 14%). Our results indicate that formalin fixation does not impede relative quantification of a phospho-site and its phosphorylation ratio in FFPE tissues. The developed workflow combining these methods opens ways to study archival FFPE tissues for phosphorylation ratio determination in proteins.

An In-Depth View of the Porcine Trabecular Meshwork Proteome.

The house swine (Sus scrofa domestica Linnaeus 1758) is an important model organism regarding the study of neurodegenerative diseases, especially ocular neuropathies such as glaucoma. This is due to the high comparability of the porcine and human eye regarding anatomy and molecular features. In the pathogenesis of glaucoma, the trabecular meshwork (TM) forms a key ocular component in terms of intraocular pressure (IOP) elevation. Thereby, functional TM abnormalities are correlated with distinct proteomic alterations. However, a detailed analysis of the TM proteome has not been realized so far. Since the porcine eye has high potential as a model system to study ocular diseases such as glaucoma, the present study focuses on the in-depth analysis of the porcine TM proteome. By use of a bottom-up (BU) mass spectrometric (MS) platform utilizing electrospray ionization liquid chromatography tandem MS (LC-ESI-MS/MS) considering database-dependent and peptide de novo sequencing, more than 3000 TM proteins were documented with high confidence (FDR < 1%). A distinct number of proteins with neuronal association were revealed. To the best to our knowledge, many of these protein species have not been reported for TM tissue before such as reelin, centlein and high abundant neuroblast differentiation-associated protein AHNAK (AHNAK). Thereby, AHNAK might play a superordinate role in the TM regarding proposed tissue involvement in barrier function. Also, a high number of secretory proteins could be identified. The generated TM proteomic landscape underlines a multifunctional character of the TM beyond representing a simple drainage system. Finally, the protein catalogue of the porcine TM provides an in-depth view of the TM molecular landscape and will serve as an important reference map in terms of glaucoma research utilizing porcine animal models, porcine TM tissues and/or cultured TM cells.

Overexpression of RNF38 facilitates TGF-\u03b2 signaling by Ubiquitinating and degrading AHNAK in hepatocellular carcinoma

BackgroundRING finger protein 38 (RNF38), a member of the RNF protein family, has just emerged as a vital driver of cancer progression. However, the oncogenic mechanisms of RNF38 remain unexplored.MethodsUsing frozen tumor tissue and tissue microarray from hepatocellular carcinoma (HCC) patients, we tried to probe the expression of RNF38 in HCC and its clinical value. Then the biological functions of RNF38 were analyzed in vivo and vitro. Stable isotope labeling with amino acids (SILAC) in cell culture and co-immunoprecipitation proteomic analyses were combined to reveal the potential mechanism of RNF38 in HCC progression.ResultsWe report that RNF38 expression was markedly higher in HCC tissues than in peritumor tissues. Correspondingly, RNF38 overexpression promoted the HCC cell migration and invasion and inhibited apoptosis both in vitro and in vivo. And elevated RNF38 expression induced HCC cell epithelial-mesenchymal transition by facilitating transforming growth factor-β (TGF-β) signaling via ubiquitinating and degrading neuroblast differentiation-associated protein (AHNAK), a well-established inhibitor of TGF-β signaling. Furthermore, AHNAK interference restored the HCC cell invasion and metastasis deprived by RNF38 downregulation. Clinically, elevated RNF38 and transforming growth factor beta receptor 1 (TGFBR1) expression was related to short overall survival (OS) and high cumulative recurrence rates in HCC patients.ConclusionsHigh levels of RNF38 promote HCC by facilitating TGF-β signaling and are a novel marker for predicting the prognosis of HCC patients and a potential therapeutic target in HCC.

Quantitative Proteomic Analysis Identifies AHNAK (Neuroblast Differentiation-associated Protein AHNAK) as a Novel Candidate Biomarker for Bladder Urothelial Carcinoma Diagnosis by Liquid-based Cytology

Cytological examination of urine is the most widely used noninvasive pathologic screen for bladder urothelial carcinoma (BLCA); however, inadequate diagnostic accuracy remains a major challenge. We performed mass spectrometry-based proteomic analysis of urine samples of ten patients with BLCA and ten paired patients with benign urothelial lesion (BUL) to identify ancillary proteomic markers for use in liquid-based cytology (LBC). A total of 4,839 proteins were identified and 112 proteins were confirmed as expressed at significantly different levels between the two groups. We also performed an independent proteomic profiling of tumor tissue samples where we identified 7,916 proteins of which 758 were differentially expressed. Cross-platform comparisons of these data with comparative mRNA expression profiles from The Cancer Genome Atlas identified four putative candidate proteins, AHNAK, EPPK1, MYH14 and OLFM4. To determine their immunocytochemical expression levels in LBC, we examined protein expression data from The Human Protein Atlas and in-house FFPE samples. We further investigated the expression of the four candidate proteins in urine cytology samples from two independent validation cohorts. These analyses revealed AHNAK as a unique intracellular protein differing in immunohistochemical expression and subcellular localization between tumor and non-tumor cells. In conclusion, this study identified a new biomarker, AHNAK, applicable to discrimination between BLCA and BUL by LBC. To our knowledge, the present study provides the first identification of a clinical biomarker for LBC based on in-depth proteomics.

A Genome-Wide Linkage Study for Chronic Obstructive Pulmonary Disease in a Dutch Genetic Isolate Identifies Novel Rare Candidate Variants.

Chronic obstructive pulmonary disease (COPD) is a complex and heritable disease, associated with multiple genetic variants. Specific familial types of COPD may be explained by rare variants, which have not been widely studied. We aimed to discover rare genetic variants underlying COPD through a genome-wide linkage scan. Affected-only analysis was performed using the 6K Illumina Linkage IV Panel in 142 cases clustered in 27 families from a genetic isolate, the Erasmus Rucphen Family (ERF) study. Potential causal variants were identified by searching for shared rare variants in the exome-sequence data of the affected members of the families contributing most to the linkage peak. The identified rare variants were then tested for association with COPD in a large meta-analysis of several cohorts. Significant evidence for linkage was observed on chromosomes 15q14–15q25 [logarithm of the odds (LOD) score = 5.52], 11p15.4–11q14.1 (LOD = 3.71) and 5q14.3–5q33.2 (LOD = 3.49). In the chromosome 15 peak, that harbors the known COPD locus for nicotinic receptors, and in the chromosome 5 peak we could not identify shared variants. In the chromosome 11 locus, we identified four rare (minor allele frequency (MAF) <0.02), predicted pathogenic, missense variants. These were shared among the affected family members. The identified variants localize to genes including neuroblast differentiation-associated protein (AHNAK), previously associated with blood biomarkers in COPD, phospholipase C Beta 3 (PLCB3), shown to increase airway hyper-responsiveness, solute carrier family 22-A11 (SLC22A11), involved in amino acid metabolism and ion transport, and metallothionein-like protein 5 (MTL5), involved in nicotinate and nicotinamide metabolism. Association of SLC22A11 and MTL5 variants were confirmed in the meta-analysis of 9,888 cases and 27,060 controls. In conclusion, we have identified novel rare variants in plausible genes related to COPD. Further studies utilizing large sample whole-genome sequencing should further confirm the associations at chromosome 11 and investigate the chromosome 15 and 5 linked regions.

Determination of the role of PAR‐associated proteins in ROS‐mediated cell death

2,3,5‐Tris‐(glutathion‐S‐yl)hydroquinone (TGHQ) is a nephrotoxic and nephrocarcinogenic metabolite of hydroquinone. TGHQ can redox cycle, generating ROS which cause DNA strand breaks, hyperactivation of poly(ADP‐ribose) polymerase‐1 (PARP‐1), increases in intracellular calcium concentration (i[Ca2+]), and cell death. PARP‐1 is a nuclear protein involved in DNA repair, transcriptional regulation and intracellular trafficking, and catalyzes the attachment of a posttranscriptional modification, consisting of multi branched ADP‐ribose polymers (PAR) on target proteins. ROS stress promotes PARP‐1 hyperactivation and elevations in i[Ca2+] which are reciprocally coupled, resulting in cell death. The molecular mechanism of this interaction is unclear. The aim of the present study was to identify TGHQ‐induced PAR‐associated proteins and their potential role in cell death. Human kidney proximal tubule cells (HK‐2) were treated with 400 μM TGHQ to induce ROS‐stress leading to PARP‐1 hyperactivation. PAR‐associated proteins were immunoprecipitated, followed by subsequent separation by SDS PAGE and proteomic analysis. A relative protein abundance analysis by spectral counts allowed us to obtain fold‐changes in protein abundance relative to control. 361 PAR‐associated proteins were significantly identified as modified by TGHQ treatment. From this cohort, 276 proteins showed an increased PAR association while 85 were decreased. Additionally, 13 targets had Gene Ontology annotations related to calcium. Of these, neuroblast differentiation‐associated protein (AHNAK), calcium homeostasis endoplasmic reticulum protein (CHERP) and general transcription factor II‐I (TFII‐I) are directly involved in modulation of i[Ca2+]. TFII‐I is a transcription factor that regulates i[Ca2+] by sequestering phospholipase C (PLC) in the cytosol. Under basal conditions free PLC binds and promotes the translocation of the transient receptor potential channel (TRPC3) to the plasma membrane leading to increases in i[Ca2+]. TFII‐I increased PAR‐association might therefore lead to calcium influx via TRPC3. Using co‐immunoprecipitation and immunodetection against TFII‐I, we confirmed increased TFII‐I PAR‐association. The effect of a TRPC3 inhibitor on TGHQ‐dependent modulation of i[Ca2+] was determined by live imaging of HK‐2 cells with the calcium sensitive dye fluo‐8 AM. Inhibition of TRPC3 promoted a delayed but higher increase in i[Ca2+]. Cellular compartmentalization of TFII‐I under TGHQ treatment was determined by cellular fractionation. TGHQ induced TFII‐I translocation from the nucleus to the cytosol as well as decreased tyrosine phosphorylation, which might have a functional effect on the transactivation potential of TFII‐I. In summary, LC‐MS/MS identified proteins with altered PARylation under ROS stress. TFII‐I is a novel PAR interacting protein which participates in signaling pathways with potential importance in TGHQ‐mediated cell death. Currently we are investigating the transcriptional and DNA repair effects of PAR‐associated TFII‐I that might be relevant to the mechanism of ROS‐dependent toxicity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Determination of Site-Specific Phosphorylation Ratios in Proteins with Targeted Mass Spectrometry.

We show that parallel reaction monitoring (PRM) can be used for exact quantification of phosphorylation ratios of proteins using stable-isotope-labeled peptides. We have compared two different PRM approaches on a digest of a U87 cell culture, namely, direct-PRM (tryptic digest measured by PRM without any further sample preparation) and TiO2-PRM (tryptic digest enriched with TiO2 cartridges, followed by PRM measurement); these approaches are compared for the following phosphorylation sites: neuroblast differentiation-associated protein (AHNAK S5480-p), calcium/calmodulin-dependent protein kinase type II subunit delta (CAMK2D T337-p), and epidermal growth factor receptor (EGFR S1166-p). A reproducible percentage of phosphorylation could be determined (CV 6-13%) using direct-PRM or TiO2-PRM. In addition, we tested the approaches in a cell culture experiment in which U87 cells were deprived of serum. As a "gold standard" we included immune precipitation of EGFR followed by PRM (IP-PRM). For EGFR (S1166) and AHNAK (S5480) a statistical significant change in the percentage of phosphorylation could be observed as a result of serum deprivation; for EGFR (S1166) this change was observed for both TiO2-PRM and IP-PRM. The presented approach has the potential to multiplex and to quantify the ratio of phosphorylation in a single analysis.

Abstract 3913: Conjugate-SELEX, a novel screening method, identifies aptamers that deliver payload to the cytosol of target cells

Abstract Introduction: Delivery of a nanoparticle payload to a specific cellular compartment is a prized goal of cancer nanomedicine. The well-known SELEX (systematic evolution of ligands by exponential enrichment) process is often used to identify aptamers that localize to target cellular compartments, but such aptamers cannot be guaranteed to traffic an attached payload nanoparticle to the target as well. We hypothesized that to find an aptamer capable of trafficking a nanoparticle to a specific cellular compartment, the screened library must consist of aptamer-nanoparticle conjugates. We therefore designed a screening procedure we call “Conjugate-SELEX”. In this process, the aptamer library is conjugated to liposomal nanoparticles, creating a nanoparticle-aptamer conjugate library for screening. At each round of Conjugate-SELEX, aptamers recovered from the desired cellular compartment are amplified, and used to create the conjugate library for the next round of Conjugate-SELEX. Negative screens against off-target cells (e.g. hepatocytes, unwanted uptake in which, causes many of the toxicities associated with nanoparticle therapeutics) facilitate the identification of an aptamer that efficiently transports payload into target cells while minimizing the uptake in off-target cells. Using fluorescently tagged particles, imaging the cells at each stage verifies the successful transport of the payload particles into the cytosol. Methods: We used conjugate-SELEX to screen for aptamers that carried an attached nanoparticle payload to the cytosol of UM-SCC-22A oral cancer cell line, while not being taken up by THLE-3 hepatocytes. IonTorrent sequencing of the remaining aptamers after each round demonstrated convergence to a small family of sequences with only one base difference between members. Mass spectrometric proteomics identified the surface receptor bound by these sequences as the neuroblast differentiation-associated protein AHNAK (desmoyokin) a ubiquitous intracellular protein with surface expression exclusively in certain epithelial cell types. Uptake studies with concertedly synthesized hit aptamer sequences showed enhanced uptake of targeted nanoparticles over controls, and increased cytotoxicity of doxorubicin in the nanoparticles over controls. Conclusions: Conjugate-SELEX is an excellent means of identifying aptamers with the ability to transport attached nanoparticle payloads to targeted cellular compartments, while minimizing off target effects. The technique also lends itself to the identification of surface receptors mediating the trafficking.[D.G., A.A., and N.V. contributed equally to this work.] Citation Format: Qingshan Mu, Akshaya Annapragada, Mayank Srivastava, Varatharasa Thiviyanathan, Xin Li, David Gorenstein, Annanth Annapragada, Nadarajah Vigneswaran. Conjugate-SELEX, a novel screening method, identifies aptamers that deliver payload to the cytosol of target cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3913.

AHNAK deficiency promotes browning and lipolysis in mice via increased responsiveness to β-adrenergic signalling.

In adipose tissue, agonists of the β3-adrenergic receptor (ADRB3) regulate lipolysis, lipid oxidation, and thermogenesis. The deficiency in the thermogenesis induced by neuroblast differentiation-associated protein AHNAK in white adipose tissue (WAT) of mice fed a high-fat diet suggests that AHNAK may stimulate energy expenditure via development of beige fat. Here, we report that AHNAK deficiency promoted browning and thermogenic gene expression in WAT but not in brown adipose tissue of mice stimulated with the ADRB3 agonist CL-316243. Consistent with the increased thermogenesis, Ahnak−/− mice exhibited an increase in energy expenditure, accompanied by elevated mitochondrial biogenesis in WAT depots in response to CL-316243. Additionally, AHNAK-deficient WAT contained more eosinophils and higher levels of type 2 cytokines (IL-4/IL-13) to promote browning of WAT in response to CL-316243. This was associated with enhanced sympathetic tone in the WAT via upregulation of adrb3 and tyrosine hydroxylase (TH) in response to β-adrenergic activation. CL-316243 activated PKA signalling and enhanced lipolysis, as evidenced by increased phosphorylation of hormone-sensitive lipase and release of free glycerol in Ahnak−/− mice compared to wild-type mice. Overall, these findings suggest an important role of AHNAK in the regulation of thermogenesis and lipolysis in WAT via β-adrenergic signalling.

Conjugate-SELEX: A High-throughput Screening of Thioaptamer-liposomal Nanoparticle Conjugates for Targeted Intracellular Delivery of Anticancer Drugs.

Patients with advanced head and neck squamous cell carcinoma receiving chemotherapy have a poor prognosis partly due to normal tissue toxicity; therefore, development of a tumor-targeted drug delivery platform to minimize collateral toxicity is a goal of cancer nanomedicine. Aptamers can achieve this purpose. While conventional Systematic Evolution of Ligands by Exponential Enrichment (SELEX) screens aptamer-only libraries and conjugates them to delivery vehicles after selection, we hypothesized that specific delivery requires screening libraries with aptamer-nanoparticle conjugates. We designed a procedure called, "Conjugate-SELEX", where liposomal nanoparticles (LNP) conjugated with aptamers is screened to identify aptamers that carried attached LNPs to the human head and neck squamous cell carcinoma cell cytosol. Aptamer-LNPs were simultaneously selected for a low affinity to human hepatocytes, minimizing hepatoxicity and LNP clearance. Post-SELEX Next Generation sequencing demonstrated convergence to a family of sequences with one base difference. Affinity pulldown and proteomics analysis identified the uptake-mediating surface receptor as the neuroblast differentiation-associated protein AHNAK (Desmoyokin), a ubiquitous intracellular protein expressed in certain epithelial cell types. Uptake studies with the lead aptamer-conjugates showed enhanced uptake and increased cytotoxicity induced by doxorubicin in cells treated with aptamer-conjugated LNPs over LNP controls. Conjugate-SELEX identifies aptamers capable of targeted cytosolic delivery of attached LNPs payload, while minimizing off-target delivery. The technique lends itself to identification of uptake-mediating surface receptors.

Proteomic Profiling of Chemotherapy Treated Triple Negative Breast Cancer Cells

ABSTRACT Aim: Triple negative breast cancer (TNBC), which represents approximately 15% of breast cancers, is defined by lack of expression of ER and PR, and lack of overexpression of HER2. Treatment options for TNBC are limited due to the lack of a clinically validated molecular target. Despite increased sensitivity to standard chemotherapy, TNBC is associated with a higher risk of relapse and poorer overall survival, than other breast cancer subtypes. The aim of this study was to profile chemotherapy-induced changes in TNBC cells, in order to identify new druggable targets that may enhance response to chemotherapy. Methods: Two TNBC cell lines, MDA-MB- 468 (cisplatin sensitive), and HCC-1143 (cisplatin resistant), were treated with cisplatin (2 µM) or docetaxel (2 nM) for 24 hours. Following treatment, proteomic profiling was performed using semi-quantitative label-free LC-MS-based proteomics analysis. Results: In cisplatin-treated HCC-1143 cells, 7 proteins with significantly (p 1.5 fold) were identified, 5 up-regulated and 2 down-regulated. In cisplatin-treated MDA-MB-468 cells, significant changes in 3 proteins were identified, with 2 increased and 1 decreased. Following docetaxel treatment, 43 altered proteins were identified in HCC1143 cells (32 increased, 11 decreased), and 3 proteins in MDA-MB-468 cells (1 increased, 2 decreased). Three proteins have been selected for further evaluation as potential targets to improve response to cisplatin and/or docetaxel. Neuroblast differentiation-associated protein (AHNAK), which has been implicated in activating PKC, is up-regulated in HCC1143 following treatment with both cisplatin (3.0-fold) and docetaxel (1.8-fold). Malate dehydrogenase (MDH2), which has been implicated in docetaxel resistance in prostate cancer, is up-regulated 2.2-fold, in docetaxel-treated HCC1143 cells. DNA-dependent protein kinase (DNA-PK) is up-regulated 1.6-fold in cisplatin-treated MDA-MB-468 cells. Previous studies suggest that DNA-PK plays a role in DNA repair following cisplatin treatment. Conclusions: In summary, we have identified three potential novel targets for TNBC which are druggable and may improve response to chemotherapy. Therapies targeting these three proteins will be evaluated, in combination with chemotherapy, in TNBC cell lines. Disclosure: J. Crown: Prof John Crown has received funding from Sanofi-Aventis and Bristol Myers Squibb for research and education activities. All other authors have declared no conflicts of interest.

Expression of Blood-Brain Barrier Antigens in Non-Brain Derived Endothelial Cells Despite Lack of Astrocyte Interactions

Endothelial cells of different vascular beds display diverse functional and morphological characteristics. Specifically, brain-derived endothelial cells exhibit specialized properties that form the blood-brain barrier, an important structure that ensures homeostasis within the neural environment. Classic proteins associated with the blood-brain barrier include neurothelin (also known as CD147), neuroblast differentiation-associated protein, glucose transporter-1 and gamma-glutamyltranspeptidase. Astrocytes are believed to be responsible for inducing the expression of blood-brain barrier proteins in brain endothelial cells. We evaluated the induction property of astrocytes in this study. Human umbilical vein endothelial cells and human dermal microvascular endothelial cells, both non-brain derived, were grown as co-cultures with human astrocytes or as monocultures for various periods. Blood-brain barrier marker mRNA levels were quantified using realtime reverse transcriptase polymerase chain reaction and protein localization was determined through immunofluorescence staining. Our results demonstrate expression of neurothelin, glucose transporter-1, and gamma-glutamyltranspeptidase mRNA in endothelial cells even in the absence of astrocytes. Co-culturing endothelial cells with astrocytes induced the expression of glucose transporter-1 and gammaResearch Article British Journal of Medicine & Medical Research, 3(1): 29-40, 2013 30 glutamyltranspeptidase but not neurothelin. Immunofluorescence staining revealed expression and localization of neuroblast differentiation-associated protein at cell membranes of non-brain derived endothelial cells. Non-brain endothelial cells also showed glucose transporter-1, gamma-glutamyltranspeptidase and neurothelinimmunoreactivity in expected subcellular compartments. These findings indicate that endothelial cells in culture express markers of blood-brain barrier and that astrocytes have differential inductive capacity depending of the endothelial cell type.

Searching for new biomarkers of bladder cancer based on proteomic analysis

Urine cytology is the gold standard for detection of bladder cancer. However, it has poor sensitivity, and currently several diagnostic markers have high false-positive rates. One of the problems with bladder cancer is high locoregional recurrence after surgery. Therefore new diagnostic markers of bladder cancer are needed. We have conducted a comprehensive study of high molecular mass (HMM) protein expression in bladder cancer tissue by means of agarose two-dimensional gel electrophoresis (agarose 2-DE) followed by the analysis of liquid chromatography tandem mass spectroscopy. We successfully identified 53 proteins from cancer tissue and 33 proteins from normal mucosa in the HMM area of the 2DE gel, and they were classified as proteins of transcription, translation or the cytoskeleton. Among them, eight differentially regulated proteins were selected as new marker candidates, and we performed experiments to validate their use as diagnostic markers. The results of Western blotting showed that these proteins except for α spectrin were significantly upregulated in bladder cancer tissue, and one of them, structural maintenance of chromosomes protein 3 (SMC3), was specifically detected in urine samples from bladder cancer patients. On the other hand, the results of Immunohistochemistry showed that expression of neuroblast differentiation-associated protein AHNAK (AHNAK) was significantly increased and that of plectin-1 was significantly decreased in the bladder cancer specimens compared with normal bladder mucosa specimens. However, no significant difference was observed in other proteins. These results suggest that AHNAK and Plectin-1 would be the potential markers for urine cytology and SMC3 is possible to be that for diagnosis marker for urinalysis.