Procollagen C-endopeptidase Research Articles

PCPE-1, a brown adipose tissue-derived cytokine, promotes obesity-induced liver fibrosis

Metabolic dysfunction-associated steatohepatitis (MASH, previously termed non-alcoholic steatohepatitis (NASH)), is a major complication of obesity that promotes fatty liver disease. MASH is characterized by progressive tissue fibrosis and sterile liver inflammation that can lead to liver cirrhosis, cancer, and death. The molecular mechanisms of fibrosis in MASH and its systemic control remain poorly understood. Here, we identified the secreted-type pro-fibrotic protein, procollagen C-endopeptidase enhancer-1 (PCPE-1), as a brown adipose tissue (BAT)-derived adipokine that promotes liver fibrosis in a murine obesity-induced MASH model. BAT-specific or systemic PCPE-1 depletion in mice ameliorated liver fibrosis, whereas, PCPE-1 gain of function in BAT enhanced hepatic fibrosis. High-calorie diet-induced ER stress increased PCPE-1 production in BAT through the activation of IRE-1/JNK/c-Fos/c-Jun signaling. Circulating PCPE-1 levels are increased in the plasma of MASH patients, suggesting a therapeutic possibility. In sum, our results uncover PCPE-1 as a novel systemic control factor of liver fibrosis.

Serum protein responses to Dietary Approaches to Stop Hypertension (DASH) and DASH-Sodium trials and associations with blood pressure changes.

The Dietary Approaches to Stop Hypertension (DASH) diet reduces blood pressure, but the mechanisms underlying DASH diet-blood pressure relations are not well understood. Proteomic measures may provide insights into the pathophysiological mechanisms through which the DASH diet reduces blood pressure. The DASH (1994-1996) and DASH-Sodium (1997-1999) trials were multicenter, randomized-controlled feeding trials. Proteomic profiling was conducted in serum collected at the end of the feeding period (DASH, N = 215; DASH-Sodium, N = 390). Multivariable linear regression models were used to identify interactions between 71 DASH diet-related proteins and changes in systolic and diastolic blood pressure. Estimates were meta-analyzed across both trials. Elastic net models were used to identify proteins that predict changes in blood pressure. Ten significant interactions were identified [systolic blood pressure: seven proteins; diastolic blood pressure: three proteins], which represented nine unique proteins. A high level of renin at the end of the feeding period was associated with greater reductions in diastolic blood pressure in individuals consuming the control than DASH diets. A high level of procollagen c-endopeptidase enhancer 1 (PCOLCE) and collagen triple helix repeat-containing protein 1 (CTHRC1) were associated with greater reductions in systolic blood pressure in individuals consuming the DASH than control diets, and with elevations in systolic blood pressure in individuals consuming the control diets (P for interaction for all tests < 0.05). Elastic net models identified six additional proteins that predicted change in blood pressure. Several novel proteins were identified that may provide some insight into the relationship between the DASH diet and blood pressure.

Pcolce2 overexpression promotes supporting cell reprogramming in the neonatal mouse cochlea.

Hair cell (HC) damage is a leading cause of sensorineural hearing loss, and in mammals supporting cells (SCs) are unable to divide and regenerate HCs after birth spontaneously. Procollagen C-endopeptidase enhancer 2 (Pcolce2), which encodes a glycoprotein that acts as a functional procollagen C protease enhancer, was screened as a candidate regulator of SC plasticity in our previous study. In the current study, we used adeno-associated virus (AAV)-ie (a newly developed adeno-associated virus that targets SCs) to overexpress Pcolce2 in SCs. AAV-Pcolce2 facilitated SC re-entry into the cell cycle both in cultured cochlear organoids and in the postnatal cochlea. In the neomycin-damaged model, regenerated HCs were detected after overexpression of Pcolce2, and these were derived from SCs that had re-entered the cell cycle. These findings reveal that Pcolce2 may serve as a therapeutic target for the regeneration of HCs to treat hearing loss.

SOX9 Promotes Collagen VI Secretion by Upregulating PCOLCE in Neurofibroma.

Neurofibromatosis type 1 (NF1) is caused by NF1 gene mutations. Patients with NF1 often have complications with tumors, such as neurofibroma. In order to investigate the pathogenesis of human neurofibroma, a systematic comparison of protein expression levels between Schwann cell-like sNF96.2 cells, which originated from malignant peripheral nerve sheath tumors (MPNST), and normal Schwann cells was performed using 4-D label-free proteomic analysis. In addition, the expression levels and localization of dysregulated proteins were confirmed using a Gene Expression Omnibus (GEO) transcriptomic dataset, Western blot analysis, and immunofluorescence labeling. The effects of SRY-box transcription factor 9 (SOX9) in the neurofibroma and surrounding microenvironment were evaluated in vivo using a tumor transplantation model. The present study observed that SOX9 and procollagen C-endopeptidase enhancer (PCOLCE) were significantly altered. NF1 mutation promoted the nuclear translocation and transcriptional activity of SOX9 in neurofibromas. SOX9 increased collagen VI secretions by enhancing the activation of PCOLCE in neurofibroma cells. These findings might provide new perspectives on the pathophysiological significance of SOX9 in neurofibromas and elucidate a novel molecular mechanism underlying neurofibromas.

Integrative analysis of transcriptome and proteome profiles in primary and recurrent glioblastoma.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor characterized by poor prognosis and high recurrence. The underlying molecular mechanism that drives tumor progression and recurrence is unclear. This study is intended to look for molecular and biological changes that play a key role in GBM recurrence. An integrative transcriptomic and proteomic analysis was performed on three primary GBM and three recurrent GBM tissues. Omics analyses were conducted using label-free quantitative proteomics and whole transcriptome sequencing. A significant difference was found between primary GBM and recurrent GBM at the transcriptional level. Similar to other omics studies of cancer, a weak overlap was observed between transcriptome and proteome, and Procollagen C-Endopeptidase Enhancer 2 (PCOLCE2) was observed to be upregulated at mRNA and protein levels. Analysis of public cancer database revealed that high expression of PCOLCE2 is associated with poor prognosis of patients with GBM and that it may be a potential prognostic indicator. Functional and environmental enrichment analyses revealed significantly altered signaling pathways related to energy metabolism, including mitochondrial ATP synthesis-coupled electron transport and oxidative phosphorylation. This study provides new insights into the recurrence process of GBM through combined transcriptomic and proteomic analyses, complementing the existing GBM transcriptomic and proteomic data and suggesting that integrated multi-omics analyses may reveal new disease features of GBM.

Macular Edema in Central Retinal Vein Occlusion Correlates With Aqueous Fibrinogen Alpha Chain.

The global protein profile of the aqueous humor has been found to correlate with the severity of retinal vascular disease. Studying the aqueous humor in central retinal vein occlusion (CRVO) with proteomic techniques may bring insights to the molecular mechanisms underlying the condition. Aqueous humor samples from treatment naïve patients with CRVO complicated by macular edema (n = 28) and age-matched controls (n = 20) were analyzed by label-free quantification liquid chromatography - tandem mass spectrometry. Best corrected visual acuity (BCVA) was measured as logMAR, and the severity of macular edema was evaluated as central retinal thickness (CRT) with optical coherence tomography. Control samples were obtained prior to cataract surgery. Significantly changed proteins were identified by a permutation-based calculation with a false discovery rate of 0.05. A total of 177 proteins were differentially expressed in CRVO. Regulated proteins were involved in complement activation, innate immune response, blood coagulation, and cell adhesion. Upregulated proteins that correlated with BCVA and CRT included fibrinogen alpha, beta, and gamma chains, fibronectin, Ig lambda-6 chain C region, Ig alpha-1 chain C region, and complement C7. Downregulated proteins that correlated negatively with BCVA, and CRT, included procollagen C-endopeptidase enhancer 1, clusterin, opticin, reelin, fibrillin-1, and cadherin-2. Monocyte differentiation antigen CD14 and lipopolysaccharide-binding protein were increased in CRVO. Fibrinogen chains, fibronectin, and immunoglobulin components correlated with BCVA and CRT, suggesting a multifactorial response. Protective anti-angiogenic proteins, including procollagen C-endopeptidase enhancer 1, clusterin, and opticin, were downregulated in CRVO and correlated negatively with BCVA and CRT.

A pan-cancer analysis of the oncogenic role of procollagen C-endopeptidase enhancer (PCOLCE) in human

There is no evidence showing that the expression of procollagen C-endopeptidase enhancer (PCOLCE) is associated with human tumors, and pan-cancer analysis is not available. Based on public databases such as the cancer genome atlas, we investigated the potential role of PCOLCE expression in 33 different human tumors. PCOLCE expression in 11 tumors was significantly correlated with tumor prognosis and was a prognostic predictor for pancreatic adenocarcinoma, thymoma and CES. We also found that PCOLCE expression correlated with the immune microenvironment of tumors and the level of cancer-associated fibroblast infiltration. PCOLCE is a potential predictor of small molecule targeted drugs and immune checkpoint inhibitors. Finally, we found by enrichment analysis that PCOLCE localizes to extracellular structures and the extracellular matrix and exerts substantial effects on tumors through the PI3K-Akt and AGE-RAGE signaling pathways. We have a preliminary and relatively comprehensive understanding of the role of PCOLCE in various tumors.

The First Pituitary Proteome Landscape From Matched Anterior and Posterior Lobes for a Better Understanding of the Pituitary Gland

To date, very few mass spectrometry (MS)–based proteomics studies are available on the anterior and posterior lobes of the pituitary. In the past, MS-based investigations have focused exclusively on the whole pituitary gland or anterior pituitary lobe. In this study, for the first time, we performed a deep MS-based analysis of five anterior and five posterior matched lobes to build the first lobe-specific pituitary proteome map, which documented 4090 proteins with isoforms, mostly mapped into chromosomes 1, 2, and 11. About 1446 differentially expressed significant proteins were identified, which were studied for lobe specificity, biological pathway enrichment, protein–protein interaction, regions specific to comparison of human brain and other neuroendocrine glands from Human Protein Atlas to identify pituitary-enriched proteins. Hormones specific to each lobe were also identified and validated with parallel reaction monitoring–based target verification. The study identified and validated hormones, growth hormone and thyroid-stimulating hormone subunit beta, exclusively to the anterior lobe whereas oxytocin–neurophysin 1 and arginine vasopressin to the posterior lobe. The study also identified proteins POU1F1 (pituitary-specific positive transcription factor 1), POMC (pro-opiomelanocortin), PCOLCE2 (procollagen C-endopeptidase enhancer 2), and NPTX2 (neuronal pentraxin-2) as pituitary-enriched proteins and was validated for their lobe specificity using parallel reaction monitoring. In addition, three uPE1 proteins, namely THEM6 (mesenchymal stem cell protein DSCD75), FSD1L (coiled-coil domain–containing protein 10), and METTL26 (methyltransferase-like 26), were identified using the NeXtProt database, and depicted tumor markers S100 proteins having high expression in the posterior lobe. In summary, the study documents the first matched anterior and posterior pituitary proteome map acting as a reference control for a better understanding of functional and nonfunctional pituitary adenomas and extrapolating the aim of the Human Proteome Project towards the investigation of the proteome of life.

Procollagen C-Endopeptidase Enhancer 2 Secreted by Tonsil-Derived Mesenchymal Stem Cells Increases the Oxidative Burst of Promyelocytic HL-60 Cells.

Simple SummaryTonsil-derived mesenchymal stem cells (TMSCs) improved the reactive oxygen species (ROS) production in human promyelocytic leukaemia cells (HL-60) differentiated into neutrophil-like cells (dHL-60). TMSC-induced enhancement of ROS generation in dHL-60 cells was different depending on the TMSC donor. Comparison of RNA-sequencing data between high and low potentiating TMSC groups for ROS generation in dHL-60 cells showed elevated expressions of four genes: secreted frizzled-related protein 4, mesenteric estrogen-dependent adipogenesis, microfibrillar associated protein 5, and procollagen C-endopeptidase enhancer 2 (PCOLCE2). Real-time PCR and Western blotting confirmed high levels of PCOLCE2 in the high potentiating TMSC group for ROS generation in dHL-60 cells. In addition, knockdown of PCOLCE2 in TMSCs reduced the enhancing efficacy of TMSCs regarding ROS generation in dHL-60 cells. Finally, treatment of recombinant PCOLCE2 protein augmented ROS production in dHL-60 cells with concomitant increases of NADPH oxidase (NOX) 3, NOX4, NOX5, and dual oxidase 2. Taken together, this study showed that PCOLCE2 levels in TMSCs could be used to select TMSCs with the high potentiating ability for ROS generation in neutrophils, and both TMSCs and PCOLCE2 may have the potential to enhance a frontline defence by increasing the efficiency of ROS generation in neutrophils.Reactive oxygen species (ROS) generated by neutrophils provide a frontline defence against invading pathogens. We investigated the supportive effect of tonsil-derived mesenchymal stem cells (TMSCs) on ROS generation from neutrophils using promyelocytic HL-60 cells. Methods: Differentiated HL-60 (dHL-60) cells were cocultured with TMSCs isolated from 25 independent donors, and ROS generation in dHL-60 cells was measured using luminescence. RNA sequencing and real-time PCR were performed to identify the candidate genes of TMSCs involved in augmenting the oxidative burst of dHL-60 cells. Transcriptome analysis of TMSCs derived from 25 independent donors revealed high levels of procollagen C-endopeptidase enhancer 2 (PCOLCE2) in TMSCs, which were highly effective in potentiating ROS generation in dHL-60 cells. In addition, PCOLCE2 knockdown in TMSCs abrogated TMSC-induced enhancement of ROS production in dHL-60 cells, indicating that TMSCs increased the oxidative burst in dHL-60 cells via PCOLCE2. Furthermore, the direct addition of recombinant PCOLCE2 protein increased ROS production in dHL-60 cells. These results suggest that PCOLCE2 secreted by TMSCs may be used as a therapeutic candidate to enhance host defences by increasing neutrophil oxidative bursts. PCOLCE2 levels in TMSCs could be used as a marker to select TMSCs exhibiting high efficacy for enhancing neutrophil oxidative bursts.

Key genes associated with prognosis and metastasis of clear cell renal cell carcinoma.

BackgroundClear cell renal cell carcinoma (ccRCC) is a tumor that frequently shows the hematogenous pathway and tends to be resistant to radiotherapy and chemotherapy. However, the exact mechanism of ccRCC metastasis remains unknown.MethodsDifferentially expressed genes (DEGs) of three gene expression profiles (GSE85258, GSE105288 and GSE22541) downloaded from the Gene Expression Omnibus (GEO) database were analyzed by GEO2R analysis, and co-expressed DEGs among the datasets were identified using a Venn drawing tool. The co-expressed DEGs were investigated using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, and hub genes were determined based on the protein-protein interaction network established by STRING. After survival analysis performed on UALCAN website, possible key genes were selected and verified in ccRCC cell lines and ccRCC tissues (n = 44). Statistical analysis was conducted using GraphPad Prism (Version 8.1.1).ResultsA total of 104 co-expressed DEGs were identified in the three datasets. Pathway analysis revealed that these genes were enriched in the extracellular matrix (ECM)–receptor interaction, protein digestion and absorption and focal adhesion. Survival analysis on 17 hub genes revealed that four key genes with a significant impact on survival: procollagen C-endopeptidase enhancer (PCOLCE), prolyl 4-hydroxylase subunit beta (P4HB), collagen type VI alpha 2 (COL6A2) and collagen type VI alpha 3 (COL6A3). Patients with higher expression of these key genes had worse survival than those with lower expression. In vitro experiments revealed that the mRNA expression levels of PCOLCE, P4HB and COL6A2 were three times higher and that of COL6A3 mRNA was 16 times higher in the metastatic ccRCC cell line Caki-1 than the corresponding primary cell line Caki-2. Immunohistochemistry revealed higher expression of the proteins encoded by these four genes in metastatic ccRCC compared with tumors from the corresponding primary sites, with statistical significance.ConclusionPCOLCE, P4HB, COL6A2 and COL6A3 are upregulated in metastatic ccRCC and might be related to poor prognosis and distant metastases.

Pcpe2, a Novel Extracellular Matrix Protein, Regulates Adipocyte SR-BI-Mediated High-Density Lipoprotein Uptake.

To investigate the role of adipocyte Pcpe2 (procollagen C-endopeptidase enhancer 2) in SR-BI (scavenger receptor class BI)-mediated HDL-C (high-density lipoprotein cholesterol) uptake and contributions to adipose lipid storage. Pcpe2, a glycoprotein devoid of intrinsic proteolytic activity, is believed to participate in extracellular protein-protein interactions, supporting SR-BI- mediated HDL-C uptake. In published studies, Pcpe2 deficiency increased the development of atherosclerosis by reducing SR-BI-mediated HDL-C catabolism, but the biological impact of this deficiency on adipocyte SR-BI-mediated HDL-C uptake is unknown. Differentiated cells from Ldlr-/-/Pcpe2-/- (Pcpe2-/-) mouse adipose tissue showed elevated SR-BI protein levels, but significantly reduced HDL-C uptake compared to Ldlr-/- (control) adipose tissue. SR-BI-mediated HDL-C uptake was restored by preincubation of cells with exogenous Pcpe2. In diet-fed mice lacking Pcpe2, significant reductions in visceral, subcutaneous, and brown adipose tissue mass were observed, despite elevations in plasma triglyceride and cholesterol concentrations. Significant positive correlations exist between adipose mass and Pcpe2 expression in both mice and humans. Overall, these findings reveal a novel and unexpected function for Pcpe2 in modulating SR-BI expression and function as it relates to adipose tissue expansion and cholesterol balance in both mice and humans.

Abstract MP33: Coordination Of Metabolic Functions By SR-BI And PCPE2 In Adipocytes

Obesity continues to be an epidemic in the United States, placing individuals at a higher risk of developing other comorbidities such as type 2 diabetes and cardiovascular disease. Adipocytes accumulate excess triglycerides and undergo hypertrophic expansion as a protective mechanism to avoid lipotoxicity. Adipocytes are also highly dynamic cells that perform many key metabolic processes including lipid and glucose metabolism. Recent collaborative studies demonstrated that SR-BI function may require the extracellular matrix protein procollagen C-endopeptidase enhancer 2 (PCPE2) in to regulate cholesterol transport in hepatocytes and adipocytes. However, the roles of SR-BI and PCPE2 in other adipocyte metabolic processes remain unclear. This knowledge gap prompted our novel hypothesis that PCPE2 facilitates SR-BI’s ability to regulate metabolic processes in adipocytes. To test our hypothesis, we have optimized an adipocyte model system where mesenchymal stem cells isolated from the outer ears of wild-type (WT), SR-BI knockout (SR-BI -/- ), and PCPE2 -/- mice were differentiated into adipocyte-like cells. We validated our adipocyte model system by demonstrating increased Oil Red O staining, mRNA expression of adipogenesis markers, and adiponectin secretion over a 9-day post-differentiation period for all three genotypes. This model system was then used to test the roles of SR-BI and PCPE2 on various metabolic functions. Preliminary data suggests that absence of SR-BI or PCPE2 slightly impairs fatty acid uptake into adipocytes despite elevated gene expression of fatty acid transporters (FABP4, CD36) compared to WT. These data further suggest that SR-BI may rely on PCPE2 to aid in transport of fatty acids. Adipocytes lacking SR-BI or PCPE2 also displayed higher levels of mitochondrial reactive oxygen species, indicating that SR-BI and PCPE2 may be important for maintaining healthy mitochondria in adipocytes. These changes in function do not appear to depend on SR-BI oligomerization, as BS 3 crosslinking studies demonstrated the presence of SR-BI oligomers in adipocytes despite the loss of PCPE2. Altogether, these data suggest that SR-BI and PCPE2 may be reliant on each other and are important for mediating metabolic processes in adipocytes.

SR‐BI &amp; PCPE2: Friends or Foes in Adipocyte Cholesterol Transport?

Obesity continues to be an epidemic in the United States, placing individuals at a higher risk of developing other comorbidities such as type 2 diabetes and cardiovascular disease. Specialized cells called adipocytes accumulate excess triglycerides and undergo hypertrophic expansion as a protective mechanism to avoid lipotoxicity and organ failure. Since adipocytes have a reduced ability to synthesize their own cholesterol, they rely heavily on circulating lipoproteins for their cholesterol supply to support membrane expansion. Scavenger receptor BI (SR-BI) is believed to be important in the adipocyte cholesterol delivery process, as this integral membrane protein is the primary receptor for high-density lipoproteins (HDL) and regulates bi-directional cholesterol transport. Collaborative studies demonstrated that SR-BI-mediated HDL-cholesteryl ester (HDL-CE) uptake in the liver requires the extracellular matrix protein procollagen C-endopeptidase enhancer 2 (PCPE2). These published studies have prompted a novel hypothesis that PCPE2 facilitates SR-BI's cholesterol transport functions to regulate adipocyte cholesterol homeostasis. To test our hypothesis, we have replicated an adipocyte model system where ear mesenchymal stem cells (EMSCs) isolated from the outer ears of mice can be differentiated into adipocytes and further used to study cholesterol transport in vitro. We validated our adipocyte model system by Oil Red O staining, mRNA expression of adipogenesis markers (PPARγ, LPL, FABP4, cEBP2α), and adiponectin secretion over the 9-day post-differentiation period in wild-type (WT), SR-BI knock-out (SR-BI-/-), and PCPE2-/- adipocytes. All three genotypes showed increased Oil Red O staining, increased mRNA expression of adipogenesis markers, and increased adiponectin secretion over the post-differentiation period. Tandem mass spectrometry analysis showed that SR-BI-/- and PCPE2-/- adipocytes had half the amount of cholesteryl esters compared to WT adipocytes, indicating that there may be disrupted cholesterol transport in the SR-BI-/- and PCPE2-/- adipocytes. Early pilot data indicate no major changes in cholesterol efflux from [3H]-cholesterol-labeled cells to HDL between WT, SR-BI-/-, and PCPE2-/- adipocytes. Experiments to assess HDL binding and HDL-CE uptake using double radiolabeled HDL particles ([125I]-HDL-[3H]-CHE) are currently ongoing. Interestingly, using an assay that assesses cholesterol distribution in membranes, we were able to show that loss of PCPE2 causes changes in cholesterol distribution within the membrane, suggesting that it may play a role in lipid raft formation. All together, we are confident that we have developed a unique in vitro adipocyte model system that can help us assess how PCPE2 may impact SR-BI-mediated cholesterol delivery.

Part 1: profiling extra cellular matrix core proteome of human fetal nucleus pulposus in search for regenerative targets

Intervertebral disc degeneration is accompanied by a loss of Extra-cellular matrix (ECM) due to an imbalance in anabolic and catabolic pathways. Identifying ECM proteins with anabolic and/or regenerative potential could be the key to developing regenerative therapies. Since human fetal discs grow and develop rapidly, studying these discs may provide valuable insights on proteins with regenerative potential. This study compares core matrisome of 9 fetal and 7 healthy adult (age 22–79) nucleus pulposus (NP), using a proteomic and bioinformatic approach. Of the 33 upregulated proteins in fetus NP’s, 20 of which were involved in ECM assembly pathways: fibromodulin, biglycan, heparan sulfate proteoglycan 2, chondroitin sulfate proteoglycan 4, procollagen C-endopeptidase enhancer and Collagen—type 1a1, 1a2, 6a1, 6a3, 11a1, 11a2, 12a1, 14a1 and 15a1. Moreover, 10 of the upregulated proteins were involved in growth pathways ‘PI3L-Akt signaling’ and ‘regulation of insulin like growth factor transport and uptake.’ Thrombospondin 1,3 and 4, tenascin C, matrilin-3, and collagen- type 1a1, 1a2, 6a1, 6a3 and 9a1. Additionally, matrillin-2 and ‘Collagen triple helix repeat containing 1’ were identified as possible regenerative proteins due to their involvement in ‘Regeneration’ and ‘tissue development’ respectively. In conclusion, the consistency of human fetal NP’s differs greatly from that of healthy adults. In view of these outcomes, the core matrisome of human fetal discs contains an abundant number of proteins that could potentially show regenerative properties, and their potential should be explored in future machinal experiments.

Enhanced anti-ageing and wound healing properties of Ficus religiosa L. bark, leaf and aerial root extract in human keratinocytes cell line (HaCaT)

Plants have played a vital role in curative and preventive healthcare. Ficus religiosa L., a medicinally important tree has been used as a traditional medicine to treat various ailments and skin diseases. However, its skin anti-ageing properties are not yet scientifically evaluated. Here we report the comparison of F. religiosa bark (FRAqB), leaf (FRAqL) and aerial-roots (FRAqAR) extracts for skin anti-ageing and wound healing properties. FRAqB, FRAqL and FRAqAR extracts preparation, cell culture, RNA isolation, real time PCR, astringent activity and wound healing assay were carried out using standard methods. Axio Observer FL-microscope and ImageJ software were used for wound healing assay analysis. The real time PCR study reveals that matrix metalloproteinase-1 (MMP) RNA was downregulated to 75% and 95% for FRAqB and FRAqAR, respectively. For procollagen C-endopeptidase enhancer 1 (PCOLCE), FRAqB and FRAqL enhanced RNA levels by 500% and 300%, respectively. FRAqB and FRAqL increased wound healing area to about 60% and FRAqAR by 50%. All three extracts demonstrated astringent activity which is significant for skin tightening. The study proves that extracts of F. religiosa are prominent candidates for pharmaceutical and cosmetic applications such as skin anti-ageing and wound healing.

Rediscovering scavenger receptor type BI: surprising new roles for the HDL receptor.

Scavenger receptor BI (SR-BI) is classically known for its role in antiatherogenic reverse cholesterol transport as it selectively takes up cholesterol esters from HDL. Here, we have highlighted recent literature that describes novel functions for SR-BI in physiology and disease. A large population-based study has revealed that patients heterozygous for the P376L mutant form of SR-BI showed significantly increased levels of plasma HDL-cholesterol and had increased risk of cardiovascular disease, demonstrating that SR-BI in humans is a significant determinant of cardiovascular disease. Furthermore, SR-BI has been shown to modulate the susceptibility to LPS-induced tissue injury and the ability of sphingosine 1 phosphate to interact with its receptor, linking SR-BI to the regulation of inflammation. In addition, important domains within the molecule (Trp-415) as well as novel regulators (procollagen C-endopeptidase enhancer protein 2) of SR-BI's selective uptake function have recently been identified. Moreover, relatively high expression levels of the SR-BI protein have been observed in a variety of cancer tissues, which is associated with a reduced overall survival rate. The HDL receptor SR-BI is a potential therapeutic target not only in the cardiovascular disease setting, but also in inflammatory conditions as well as in cancer.

Proteomic analysis of cerebrospinal fluid from children with central nervous system tumors identifies candidate proteins relating to tumor metastatic spread.

Central nervous system (CNS) tumors are the most common solid tumors in childhood. Since the sensitivity of combined cerebrospinal fluid (CSF) cytology and radiological neuroimaging in detecting meningeal metastases remains relatively low, we sought to characterize the CSF proteome of patients with CSF tumors to identify biomarkers predictive of metastatic spread. CSF samples from 27 children with brain tumors and 13 controls (extra-CNS non-Hodgkin lymphoma) were processed using core-shell hydrogel nanoparticles, and analyzed with reverse-phase liquid chromatography/electrospray tandem mass spectrometry (LC-MS/MS). Candidate proteins were identified with Fisher's exact test and/or a univariate logistic regression model. Reverse phase protein array (RPPA), Western blot (WB), and ELISA were used in the training set and in an independent set of CFS samples (60 cases, 14 controls) to validate our discovery findings. Among the 558 non-redundant proteins identified by LC-MS/MS, 147 were missing from the CSF database at http://www.biosino.org. Fourteen of the 26 final top-candidate proteins were chosen for validation with WB, RPPA and ELISA methods. Six proteins (type 1 collagen, insulin-like growth factor binding protein 4, procollagen C-endopeptidase enhancer 1, glial cell-line derived neurotrophic factor receptor α2, inter-alpha-trypsin inhibitor heavy chain 4, neural proliferation and differentiation control protein-1) revealed the ability to discriminate metastatic cases from controls. Combining a unique dataset of CSFs from pediatric CNS tumors with a novel enabling nanotechnology led us to identify CSF proteins potentially related to metastatic status.

Abstract 191: Adipocyte Procollagen C-endopeptidase Enhancer Protein 2 (PCPE2) Influences Lipid Metabolism Secondary to Changes in Expression and Function of Scavenger Receptor Class B Member 1 (SR-BI)

Scavenger receptor class B member 1 (SR-BI) functions as a HDL receptor and stimulates reverse cholesterol transport (RCT) via selective uptake of HDL cholesterol esters (HDL-CE). Procollagen c-endopeptidase enhancer protein 2 (PCPE2) is an extracellular matrix glycoprotein encoded by the PCOLCE2 gene and most highly expressed in adipose tissue, heart, and aorta. PCPE2 has been suggested to be atheroprotective secondary to mechanisms involved in lipid metabolism. Our lab previously reported that SR-BI mediated cholesterol ester uptake is reduced in the absence of PCPE2 despite its increased expression levels. Subsequent HDL turnover studies suggested an overall reduction in catabolism, consistent with larger HDL particles observed, is responsible for the paradox. More recently, human data from the TwinsUK study showed strong correlations between PCOLCE2 and adipose tissue distribution with BMI included as a covariate. Furthermore, despite similar overall body weights, our lab observed a significant reduction in visceral fat pad development in PCPE2-deficient mice fed a Western diet for 25 weeks when compared to wild type mice. In addition, our lab has generated PCPE2 knockout cell lines from murine preadipocyte 3T3-L1 cells (PCPE2 -/- 3T3-L1) and Chinese hamster ovary (CHO) cells using CRISPR-Cas9 and siRNA technology, respectfully. In addition to both cell lines showing increased SR-BI expression, differentiated PCPE2 -/- 3T3-L1 cells have impaired lipid droplet formation. To elucidate the relationship between PCPE2 and SR-BI function and their effects on adipose tissue development, we performed RNA sequencing on visceral adipose tissue from PCPE2-deficient male mice fed a Western diet for 12 weeks. Genes exhibiting significant expression alterations (FDR &lt;0.026) compared to wild type were further analyzed via Ingenuity Pathway Analysis (IPA). Differential expression analysis of diseases and functions related to changes in SR-BI expression highlighted permutations of a number of lipid modulating genes. Overall, changes in SR-BI related metabolic pathways secondary to PCPE2 deficiency appear to play a crucial role linking SR-BI mediated cholesterol ester uptake to adipocyte biology.

Abstract 558: PCPE2 Promotes Adipocyte Maturation via Regulation of SR-BI Activity

Procollagen C-endopeptidase enhancer protein 2 (PCPE2) is an enhancer protein that enhances the cleavage of the C-termini of procollagen by bone morphogenetic protein 1. But the function of PCPE2 is not limited to collagen maturation. Recently our lab reported that PCPE2 increased HDL-associated cholesteryl ester uptake via scavenger receptor class B type 1 (SR-BI), an HDL cholesterol receptor highly expressed in adipose tissue. Interestingly, TwinsUK study in human provided data showing that PCPE2 mRNA is highly correlated with adipose tissue distribution. Further, our lab observed a reduced size of visceral fat pad in PCPE2 deficient mice despite its indifference in body weight compared to the wild type. To study the molecular and cellular mechanisms of how PCPE2 regulates SR-BI function and how it affects adipose tissue formation, we generated PCPE2 knockout (PCPE2 -/- ) cell line from murine preadipocyte 3T3-L1 cell using CRISPR-Cas9 technology. Induction of 3T3-L1 cell to differentiate into mature adipocyte increases the expression of both PCPE2 and SR-BI hand in hand around 3 fold, which parallels the process of lipid droplet formation. Immunofluorescence studies showed that PCPE2 is distributed all over the cell in mature adipocyte, while SR-BI is mostly distributed along the cytoplasmic and lipid droplet membranes, in addition to the perinuclear region. More interestingly, PCPE2 -/- 3T3-L1 cell shows an impairment in lipid droplet formation during the induction of differentiation, with less than 10% of cells generating lipid droplets, and the size of lipid droplet being only about 50% of that in wild type. Although PCPE2 -/- 3T3-L1 cell expresses SR-BI at a higher level compared with wild type cell, nearly 50% of the SR-BI in wild type cell are in homodimer structure while PCPE2 -/- 3T3-L1 cell has almost none SR-BI multimer, indicating that SR-BI in PCPE2 -/- 3T3-L1 cell is inactive. Taken together, we concluded that PCPE2 plays a critical role in keeping SR-BI in active conformation, which, in turn, controls adipocyte maturation. Whether this effect is associated with collagen maturation will be assessed in our future studies.

Influence of WFIKKN1 on BMP1-mediated activation of latent myostatin.

Furin: EC 3.4.21.75; BMP1, bone morphogentic protein 1 or procollagen C-endopeptidase: EC 3.4.24.19.