Total Membrane Fraction Research Articles

PD-L1 Up-Regulation in Prostate Cancer Cells by Porphyromonas gingivalis.

Chronic inflammation is known to contribute to various human cancers. Porphyromonas gingivalis (P. gingivalis), is a gram-negative oral keystone pathogen that may cause severe periodontitis and expresses several virulence factors to affect the host immune system. Periodontitis is a chronic infectious disease that while progression, may cause loss of attachment and destruction of the tooth supporting tissues. Prostate cancer is one of the most common malignancies in men. Increasing evidence links periodontitis with prostate cancer, however the mechanisms explaining this relationship remain unclear. The aim of this study was to investigate the expression and signaling pathway of programmed death ligand 1 (PD-L1) in a prostate cancer cell line after infection with P. gingivalis and stimulation with P. gingivalis components to reveal the mechanism of tumor-induced immune evasion associated with bacterial infection in the tumor environment. Prostate cancer cells were infected with different concentrations of viable P. gingivalis and treated with different concentrations of heat-killed P. gingivalis and P. gingivalis cell components, including the total membrane fraction, inner membrane fraction, outer membrane fraction, cytosolic fraction and peptidoglycan (PGN). Chemical inhibitors were used to block different important molecules of signaling pathways to assess the participating signal transduction mechanisms. PD-L1 expression was detected by Western blot after 24 h of infection. PD-L1 was demonstrated to be upregulated in prostate cancer cells after infection with viable and with heat-killed P. gingivalis membrane fractions. Also isolated PGN induced PD-L1 up-regulation. The upregulation was mediated by the NOD1/NOD2 signaling pathway. No upregulation could be detected after treatment of the cells with P. gingivalis lipopolysaccharide (LPS). These results indicate, that chronic inflammatory disease can contribute to tumor immune evasion by modifying the tumor microenvironment. Thus, chronic infection possibly plays an essential role in the immune response and may promote the development and progression of prostate cancer.

Interindividual Variability and Differential Tissue Abundance of Mitochondrial Amidoxime Reducing Component Enzymes in Humans.

Mitochondrial amidoxime-reducing component (mARC) enzymes are molybdenum-containing proteins that metabolize a number of endobiotics and xenobiotics. The interindividual variability and differential tissue abundance of mARC1 and mARC2 were quantified using targeted proteomics in three types of tissue fractions: 1) pediatric liver tissue homogenates, 2) total membrane fraction of the paired liver and kidney samples from pediatric and adult donors, and 3) pooled S9 fractions of the liver, intestine, kidney, lung, and heart. The absolute levels of mARC1 and mARC2 in the pediatric liver homogenate were 40.08 ± 4.26 and 24.58 ± 4.02 pmol/mg homogenate protein, respectively, and were independent of age and sex. In the total membrane fraction of the paired liver and kidney samples, the abundance of hepatic mARC1 and mARC2 was comparable, whereas mARC2 abundance in the kidney was approximately 9-fold higher in comparison with mARC1. The analysis of the third set of samples (i.e., S9 fraction) revealed that mARC1 abundance in the kidney, intestine, and lung was 5- to 13-fold lower than the liver S9 abundance, whereas mARC2 abundance was approximately 3- and 16-fold lower in the intestine and lung than the liver S9, respectively. In contrast, the kidney mARC2 abundance in the S9 fraction was approximately 2.5-fold higher as compared with the hepatic mARC2 abundance. The abundance of mARC enzymes in the heart was below the limit of quantification (∼0.6 pmol/mg protein). The mARC enzyme abundance data presented here can be used to develop physiologically based pharmacokinetic models for the prediction of in vivo pharmacokinetics of mARC substrates. SIGNIFICANCE STATEMENT: A precise targeted quantitative proteomics method was developed and applied to quantify newly discovered drug-metabolizing enzymes, mARC1 and mARC2, in pediatric and adult tissue samples. The data suggest that mARC enzymes are ubiquitously expressed in an isoform-specific manner in the human liver, kidney, intestine, and lung, and the enzyme abundance is not associated with age and sex. These data are important for developing physiologically based pharmacokinetic models for the prediction of in vivo pharmacokinetics of mARC substrates.

Quantitation of Plasma Membrane Drug Transporters in Kidney Tissue and Cell Lines Using a Novel Proteomic Approach Enabled a Prospective Prediction of Metformin Disposition.

The successful prospective incorporation of in vitro transporter kinetics in physiologically based pharmacokinetic (PBPK) models to describe drug disposition remains challenging. Although determination of scaling factors to extrapolate in vitro to in vivo transporter kinetics has been facilitated by quantitative proteomics, no robust assessment comparing membrane recoveries between different cells/tissues has been made. HEK293 cells overexpressing OCT2, MATE1, and MATE2K or human kidney cortex were homogenized and centrifuged to obtain the total membrane fractions, which were subsequently subjected to liquid-liquid extraction followed by centrifugation and precipitation to isolate plasma membrane fractions. Plasma membrane recoveries determined by quantitation of the marker Na+/K+-ATPase in lysate and plasma membrane fractions were ≤20% but within 3-fold across different cells and tissues. A separate study demonstrated that recoveries are comparable between basolateral and apical membranes of renal proximal tubules, as measured by Na+/K+-ATPase and γ-glutamyl transpeptidase 1, respectively. The plasma membrane expression of OCT2, MATE1, and MATE2K was quantified and relative expression factors (REFs) were determined as the ratio between the tissue and cell concentrations. Corrections using plasma membrane recovery had minimal impact on REF values (<2-fold). In vitro transporter kinetics of metformin were extrapolated to in vivo using the corresponding REFs in a PBPK model. The simulated metformin exposures were within 2-fold of clinical exposure. These results demonstrate that transporter REFs based on plasma membrane expression enable a prediction of transporter-mediated drug disposition. Such REFs may be estimated without the correction of plasma membrane recovery when the same procedure is applied between different matrices. SIGNIFICANCE STATEMENT: Transporter REFs based on plasma membrane expression enable in vitro-in vivo extrapolation of transporter kinetics. Plasma membrane recoveries as determined by the quantification of sodium-potassium adenosine triphosphatase were comparable between the in vitro and in vivo systems used in the present study, and therefore had minimal impact on the transporter REF values.

Porphyromonas gingivalis induced up-regulation of PD-L1 in colon carcinoma cells.

Programmed death-ligand-1 (PD-L1) is a ligand for programmed death receptor (PD-1) that plays a major role in cell-mediated immune response; it regulates T-cell activation and regulates survival and functions of activated T cells. Expression of PD-L1 can induce chronic inflammation and activate mechanisms of immune evasion. PD-L1 is expressed in most of human carcinomas. Porphyromonas gingivalis (P. gingivalis) is a major keystone pathogen in periodontitis that invade host cells and disposes a variety of virulence factors. The aim of the present study was to clarify the signaling pathway of P. gingivalis molecules that induce PD-L1 up-regulation in colon carcinoma cells. Additionally, it was investigated which components of P. gingivalis are responsible for PD-L1 induction. Colon cancer cells (CL-11) were stimulated with total membrane (TM) fractions, peptidoglycans (PDGs) and viable P. gingivalis bacteria. Seven signaling molecule inhibitors were used: receptor-interacting serine/threonine-protein kinase 2 (RIP2) tyrosine kinase inhibitor, nucleotide-binding oligomerization domain (NOD)-like receptor 1&2 inhibitor, NOD-like receptor, nuclear factor kappa B inhibitor, c-Jun N-terminal kinases inhibitor, mitogen-activated protein/extracellular signal-regulated kinase inhibitor, mitogen activated kinase (MAPK) inhibitor. PD-L1 protein expression was examined by western blot analysis and quantitative real time PCR. It was demonstrated that the TM fraction and PDG induced up-regulation of PD-L1 expression in colon cancer cells. In conclusion, the results of this study suggest that PDG of P. gingivalis plays a major role in PD-L1 up-regulation in colon cancer cells. In addition, the mechanism of PD-L1 up-regulation depends on NOD 1 and NOD 2 and involves activation of RIP2 and MAPK signaling pathways.

PRODUCTION OF POLYCLONAL ANTIBODIES FOR DETECTING HUMAN TYPE 2 TRANSGLUTAMINASE VARIANTS IN COLON CANCER

Type 2 transglutaminase (TGM2) is a multifunctional ubiquitous protein, involving in protein cross-linking, apoptosis, and cell differentiation. Recently, some reports have suggested that TGM2 expression is a potential prognostic marker, and often associates with advanced stages of disease, metastatic spread, as well as drug resistance in many cancer cell lines although its primary function is unknown. To elucidate the role of TGM2 in cancer, the expression profile of the TGM2 need to be examined. In this study, new polyclonal antibodies detecting four TGM2 variants encoding protein from ENSEMBL database are produced and their specificities are confirmed by western blot analysis with E.coli overexpressing TGM2-002 protein, HEK293T cells overexpressing TGM2-S protein and human colon cancer samples. Western blot data showed that these antibodies could detect not only TGM2-002 in E. coli overexpressing TGM2-002 but also smaller molecular weight protein (about 62 kDa) in HEK293T overexpressing TGM2-S cells which was further confirmed by MALDI-TOF analysis. We found that both TGM2-1 and TGM2-4 antibodies could detect the full length TGM2-002 protein in colon cancer samples. Furthermore, in normal sample, we found that majority of TGM2-002 protein existed in membrane fraction but not in total lysate whereas TGM2-002 protein was found in both total lysate and membrane fraction in colon cancer samples.

P-Rex1 Mediates Glucose-Stimulated Rac1 Activation and Insulin Secretion in Pancreatic β-Cells.

Despite the published evidence implicating phosphoinositide 3-kinase (PI3-kinase) in the regulation of islet function, limited information is available on the putative contributory roles of its downstream signaling steps, including the phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 1 (P-Rex1) signaling pathway in the islet β-cell. Therefore, we investigated potential roles for P-Rex1 in glucose-stimulated Rac1 activation and insulin secretion in insulin-secreting (INS-1 832/13) β-cells. Glucose-stimulated Insulin secretion (GSIS) was quantified by ELISA. Expression of endogenous P-Rex1 and RhoG was suppressed by siRNA transfection using the DharmaFect1 reagent. Total membrane and cytosolic fractions were isolated using the Mem-PER Plus Membrane Extraction Kit. The degree of activation of Rac1 was determined by the pull-down assay. P-Rex1 is expressed in INS-1 832/13 cells, normal rat islets and human islets. siRNA-mediated knockdown of P-Rex1 attenuated glucose-induced Rac1 activation, membrane association and insulin secretion. RhoG, which has been implicated in PI3-kinase-mediated Rac1 activation in other cell types, appears not to contribute to GSIS since the siRNA-mediated knockdown of RhoG failed to exert significant effects on GSIS. LY294002, a known inhibitor of PI3-kinase, potentiated GSIS without affecting glucose-induced Rac1 activation. Based on these findings, we conclude that P-Rex1 plays a novel regulatory role in glucose-induced Rac1 activation and insulin secretion.

Elevating Insulin Signaling Using a Constitutively Active Insulin Receptor Increases Glucose Metabolism and Expression of GLUT3 in Hippocampal Neurons.

Insulin signaling is an integral component of healthy brain function, with evidence of positive insulin-mediated alterations in synaptic integrity, cerebral blood flow, inflammation, and memory. However, the specific pathways targeted by this peptide remain unclear. Previously, our lab used a molecular approach to characterize the impact of insulin signaling on voltage-gated calcium channels and has also shown that acute insulin administration reduces calcium-induced calcium release in hippocampal neurons. Here, we explore the relationship between insulin receptor signaling and glucose metabolism using similar methods. Mixed, primary hippocampal cultures were infected with either a control lentivirus or one containing a constitutively active human insulin receptor (IRβ). 2-NBDG imaging was used to obtain indirect measures of glucose uptake and utilization. Other outcome measures include Western immunoblots of GLUT3 and GLUT4 on total membrane and cytosolic subcellular fractions. Glucose imaging data indicate that neurons expressing IRβ show significant elevations in uptake and rates of utilization compared to controls. As expected, astrocytes did not respond to the IRβ treatment. Quantification of Western immunoblots show that IRβ is associated with significant elevations in GLUT3 expression, particularly in the total membrane subcellular fraction, but did not alter GLUT4 expression in either fraction. Our work suggests that insulin plays a significant role in mediating neuronal glucose metabolism, potentially through an upregulation in the expression of GLUT3. This provides further evidence for a potential therapeutic mechanism underlying the beneficial impact of intranasal insulin in the clinic.

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii.

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to assess the purity of the bilayers. The OM has an increased overall density compared to the inner membrane, largely due to the presence of lipooligosaccharides (LOS) and lipopolysaccharides (LPS) within the outer leaflet. LOS and LPS molecules are amphipathic glycolipids that have a similar structure, which consists of a lipid-A disaccharolipid and core-oligosaccharide substituent. However, only LPS molecules are decorated with a third subunit known as the O-polysaccharide, or O-antigen. The type and amount of glycolipids present will impact an organism's OM density. Therefore, we tested whether the membranes of bacteria with varied glycolipid content could be similarly isolated using our technique. For the LPS-producing organisms, Salmonella enterica serovar Typhimurium and Escherichia coli, the membranes were easily isolated and the LPS O-antigen moiety did not impact bilayer partitioning. Acinetobacter baumannii produces LOS molecules, which have a similar mass to O-antigen deficient LPS molecules; however, the membranes of these microbes could not initially be separated. We reasoned that the OM of A. baumannii was less dense than that of Enterobacteriaceae, so the sucrose gradient was adjusted and the membranes were isolated. The technique can therefore be adapted and modified for use with other organisms.

Trafficking of the Prorenin Receptor in Endothelial Cells

Diabetic patients are at higher risk for hypertension and cardiovascular mortality than their normotensive, nondiabetic counterparts. Diabetic patients also have increased circulating prorenin which can bind to its receptor (PRR) to cause activation of the renin‐angiotensin system and fibrosis. We hypothesized that diabetes induces dysregulation of the endothelial PRR to exacerbate cardiovascular damage. Mouse pancreatic endothelial cells (MS1) were cultured and exposed to high glucose or low calcium (Ca2+) conditions for one hour. Cells were fixed for immunocytochemistry or fractionated and immunoblotted for PRR. Treatment with 25 mM glucose or 25 mM mannitol for one hour did not impact full length PRR expression or localization in the total membrane fraction. When Ca2+ was removed from the media, full length PRR increased in the cytosolic fraction by 3.6‐fold (P=0.022) compared to control. Restoration of physiological Ca2+ returned PRR expression to control levels in the cytosolic fraction, while neither sPRR nor full length PRR were altered in the total membrane fraction. Confocal imaging of fixed cells suggested that PRR in the total membrane fraction may be encapsulated in vesicles. These data indicate that Ca2+ may be an important regulator of endothelial PRR trafficking, possibly via a Ca2+‐dependent PRR cleavage pathway. Ongoing studies will establish the relationship between Ca2+ and sPRR/PRR and determine the impact of PRR trafficking on endothelial dysfunction. These studies will determine whether targeting of PRR can lower the risk of cardiovascular disease in patients with diabetes.Support or Funding InformationTulane Center for Engaged Learning and Technology, NIH HL133619, DK104375

A simple protocol for the isolation of proteorhodopsins of the dinoflagellate Oxyrrhis marina.

For the first time, native proteorhodopsins of the marine dinoflagellate Oxyrrhis marina were isolated. Total cell membrane fractions were minced in a bead beater and solubilized with the detergent Triton X-100. Subsequent sucrose density gradient centrifugation resulted in three or four red-colored bands. Nonsolubilized, but still red colored, membranes sedimented at the bottom. For each of these bands, absorbance maxima were registered at approximately 514-516 nm with shoulders toward shorter wavelengths (470-490 nm). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the uppermost band represented free retinal chromophore, as it contained no protein. The other bands were almost pure proteorhodopsin fractions as the banding patterns showed one major protein of 25 kDa. Tryptic, in-gel digestion of the 25 kDa proteins and of faint protein bands above and below 25 kDa was followed by mass spectrometry, confirming these protein bands to consist, nearly exclusively, proteorhodopsins. Only single peptides of few other proteins were detected. In total, at least seven predicted proteorhodopsin protein sequences were experimentally verified.

Unbiased Thiol-Labeling and Top-Down Proteomic Analyses Implicate Multiple Proteins in the Late Steps of Regulated Secretion.

Regulated exocytosis enables temporal and spatial control over the secretion of biologically active compounds; however, the mechanism by which Ca2+ modulates different stages of exocytosis is still poorly understood. For an unbiased, top-down proteomic approach, select thiol- reactive reagents were used to investigate this process in release-ready native secretory vesicles. We previously characterized a biphasic effect of these reagents on Ca2+-triggered exocytosis: low doses potentiated Ca2+ sensitivity, whereas high doses inhibited Ca2+ sensitivity and extent of vesicle fusion. Capitalizing on this novel potentiating effect, we have now identified fluorescent thiol- reactive reagents producing the same effects: Lucifer yellow iodoacetamide, monobromobimane, and dibromobimane. Top-down proteomic analyses of fluorescently labeled proteins from total and cholesterol-enriched vesicle membrane fractions using two-dimensional gel electrophoresis coupled with mass spectrometry identified several candidate targets, some of which have been previously linked to the late steps of regulated exocytosis and some of which are novel. Initial validation studies indicate that Rab proteins are involved in the modulation of Ca2+ sensitivity, and thus the efficiency of membrane fusion, which may, in part, be linked to their previously identified upstream roles in vesicle docking.

Major glucuronide metabolites of testosterone are primarily transported by MRP2 and MRP3 in human liver, intestine and kidney

Testosterone glucuronide (TG), androsterone glucuronide (AG), etiocholanolone glucuronide (EtioG) and dihydrotestosterone glucuronide (DHTG) are the major metabolites of testosterone (T), which are excreted in urine and bile. Glucuronides can be deconjugated to active androgen in gut lumen after biliary excretion, which in turn can affect physiological levels of androgens. The goal of this study was to quantitatively characterize the mechanisms by which TG, AG, EtioG and DHTG are eliminated from liver, intestine, and kidney utilizing relative expression factor (REF) approach. Using vesicular transport assay with recombinant human MRP2, MRP3, MRP4, MDR1 and BCRP, we first identified that TG, AG, EtioG, and DHTG were primarily substrates of MRP2 and MRP3, although lower levels of transport were also observed with MDR1 and BCRP vesicles. The transport kinetic analyses revealed higher intrinsic clearances of TG by MRP2 and MRP3 as compared to that of DHTG, AG, and EtioG. MRP3 exhibited higher affinity for the transport of the studied glucuronides than MRP2. We next quantified the protein abundances of these efflux transporters in vesicles and compared the same with pooled total membrane fractions isolated from human tissues by quantitative LC–MS/MS proteomics. The fractional contribution of individual transporters (ft) was estimated by proteomics-based physiological scaling factors, i.e., transporter abundance in whole tissue versus vesicles, and corrected for inside-out vesicles (determined by 5′-nucleotidase assay). The glucuronides of inactive androgens, AG and EtioG were preferentially transported by MRP3, whereas the glucuronides of active androgens, TG and DHTG were mainly transported by MRP2 in liver. Efflux by bile canalicular transport may indicate the potential role of enterohepatic recirculation in regulating the circulating active androgens after deconjugation in the gut. In intestine, MRP3 possibly contributes most to the efflux of these glucuronides. In kidney, all studied glucuronides seemed to be preferentially effluxed by MRP2 and MDR1 (for EtioG). These REF based analysis need to be confirmed with in vivo findings. Overall, characterization of the efflux mechanisms of T glucuronide metabolites is important for predicting the androgen disposition and interindividual variability, including drug-androgen interaction in humans. The mechanistic data can be extrapolated to other androgen relevant organs (e.g. prostate, testis and placenta) by integrating these data with quantitative tissue proteomics data.

IL‐15 Upregulates GLUT1 while Reducing CPT1 in Cardiomyocytes

Obesity is an exponentially growing epidemic in the United States and is a leading contributor of cardiovascular disease (CVD). Maintenance of lifestyle interventions to prevent and treat obesity, such as diet and exercise, is a challenge. In this regard, targeting the molecular mediators of obesity has become a focus to prevent CVD disease progression. In an undiseased state, the cardiac muscle relies primarily on oxidative processes for energy production. In some cases, CVD can lead to upregulation of glycogenic, rather than oxidative, processes to produce ATP for muscle contraction. The myokine interleukin‐15 (IL‐15) is secreted by skeletal muscle during exercise and has been shown to reduce obesity and related disorders. However, the effects of IL‐15 signaling in cardiomyocytes (CM) remains largely unknown. The objective of the current study was to determine if IL‐15 regulates glucose and/or fatty acid (FA) metabolism in CM. Differentiated CM were treated with 100 ng/mL of IL‐15 for 24 hours. Protein was harvested, from either IL‐15 or vehicle treated cells, and isolated into total, mitochondrial and plasma membrane (PM) fractions. The protein concentration was quantified utilizing a Bradford assay. Subsequently, protein analysis, via Western blotting, was performed. Fifteen micrograms of protein was loaded onto 4–20% polyacrylamide gels for electrophoresis. The proteins were then transferred onto a PVDF membrane. Primary antibodies against GLUT1, GLUT4, and CPT1 (1:1000) were used, followed by secondary antibodies (1:2000). GLUT1 and GLUT4 were analyzed in both total cell fractions and PM fractions, while CPT1 levels were assessed in mitochondrial fractions. The membranes were subjected to enhanced chemiluminescence, imaged and quantified with ImageJ software. A t‐test (P&lt;0.05) was used to determine if IL‐15 had any significant effects on the protein levels of the aforementioned proteins. IL‐15 did not alter total GLUT4, PM GLUT4 or PM GLUT1 (P&gt;0.05). Conversely, IL‐15 treatment resulted in a 367% increase in total GLUT1 protein levels when compared to the vehicle control cells (P&lt;0.05). Of note, IL‐15 decreased mitochondrial CPT1 by nearly 50% (P&lt;0.05). Our data indicate that IL‐15 increases the protein concentration of one of the glucose transporters, GLUT1, and decreases the protein concentration of the mitochondrial FA transporter, CPT1, in CM. Thus, IL‐15 may be responsible for promoting a shift in CM metabolism from FA to glucose utilization. Additional studies are warranted to confirm this proposed shift in metabolism by studying glucose uptake and beta‐oxidation activity in CM. Further, to understand if an IL‐15‐mediated shift in metabolism is beneficial to CM, further studies aimed at assessing cardiac muscle function are required. Taken together, if IL‐15 holds the potential as a treatment for obesity, examination of its functions in various tissues is required.Support or Funding InformationChapman University Crean College of Health and Behavioral Sciences Summer Undergraduate Research Fellowship; American Heart Association 16SDG30680003This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Kinetics of dextromethorphan-O-demethylase activity and distribution of CYP2D in four commonly-used subcellular fractions of rat brain

The purpose of this study was to compare the enzymatic kinetics and distribution of cytochrome P450 2D (CYP2D) among different rat brain subcellular fractions.Rat brains were used to prepare total membrane, crude mitochondrial, purified mitochondrial, and microsomal fractions, in addition to total homogenate. Michaelis–Menten kinetics of the brain CYP2D activity was estimated based on the conversion of dextromethorphan (DXM) to dextrorphan using UPLC-MS/MS. Protein levels of CYP2D and subcellular markers were determined by Western blot.Microsomal CYP2D exhibited high affinity and low capacity, compared with the mitochondrial CYP2D that had a much lower (∼50-fold) affinity but a higher (∼six-fold) capacity. The apparent CYP2D affinity and capacity of the crude mitochondria were in between those of the microsomes and purified mitochondria. Additionally, the CYP2D activity in the whole homogenate was much higher than that in the total membranes at higher DXM concentrations. A CYP2D immune-reactive band in the brain mitochondria appeared at a lower MW but had a much higher intensity than that in the microsomes.Mitochondrial brain CYP2D has a much higher capacity than its microsomal counterpart. Additionally, brain homogenate is more representative of the overall CYP2D activity than the widely-used total membrane fraction.

Targeted LC-MS/MS Proteomics-Based Strategy To Characterize in Vitro Models Used in Drug Metabolism and Transport Studies.

Subcellular fractionation of tissue homogenate provides enriched in vitro models (e.g., microsomes, cytosol, or membranes), which are routinely used in the drug metabolism or transporter activity and protein abundance studies. However, batch-to-batch or interlaboratory variability in the recovery, enrichment, and purity of the subcellular fractions can affect performance of in vitro models leading to inaccurate in vitro to in vivo extrapolation (IVIVE) of drug clearance. To evaluate the quality of subcellular fractions, we developed a simple, targeted, and sensitive LC-MS/MS proteomics-based strategy, which relies on determination of protein markers of various cellular organelles, i.e., plasma membrane, cytosol, nuclei, mitochondria, endoplasmic reticulum (ER), lysosomes, peroxisomes, cytoskeleton, and exosomes. Application of the quantitative proteomics method confirmed a significant effect of processing variables (i.e., homogenization method and centrifugation speed) on the recovery, enrichment, and purity of isolated proteins in microsomes and cytosol. Particularly, markers of endoplasmic reticulum lumen and mitochondrial lumen were enriched in the cytosolic fractions as a result of their release during homogenization. Similarly, the relative recovery and composition of the total membrane fraction isolated from cell vs tissue samples was quantitatively different and should be considered in IVIVE. Further, analysis of exosomes isolated from sandwich-cultured hepatocyte media showed the effect of culture duration on compositions of purified exosomes. Therefore, the quantitative proteomics-based strategy developed here can be applied for efficient and simultaneous determination of multiple protein markers of various cellular organelles when compared to antibody- or activity-based assays and can be used for quality control of subcellular fractionation procedures including in vitro model development for drug metabolism and transport studies.

Abstract 529: Asporin - Extracellular Matrix Protein Mitigates Pathological Remodeling

The cardiac extracellular matrix is a complex network of proteins and plays a key role in cardiac remodeling after myocardial infarction. One of the extracellular matrix proteins, asporin, is a member of the SLRP family and inhibits TGFβ signaling. However, very little is known about the function and regulation of asporin in cardiac remodeling. Using bioinformatics approach, microarray data analysis from publicly available datasets identified asporin as a top candidate showing an increase in left ventricle tissue with ischemic cardiomyopathy as compared to control donor hearts. To confirm, we detected an increase in asporin expression in paired heart biopsies taken before and after cardiopulmonary bypass surgery by western blot analysis. Our group showed that glucagon like receptor 1 agonist (GLP-1Ra) is beneficial for cardiac remodeling and prevents fibrosis. In a permanent coronary artery ligation model, we found increased extracellular asporin expression after 30 days in border zone and scar area with GLP-1Ra treatment compared to vehicle-treatment. To further elucidate the mechanism of asporin secretion, we used H9c2 cells as well as left ventricle tissue from mice and found that asporin was predominantly present in the heavy membrane enriched fraction (8,000xg pellet from postnuclear supernatant). Sequence analysis of asporin protein using SecretomeP 2.0 server predicted asporin might use the non-conventional form of secretion (secretory autophagy). Differentiation of H9c2 cells is accompanied by autophagy and mitochondrial biogenesis, and we found that the abundance of asporin in total lysate and heavy membrane fraction decreased suggesting it could be released via secretory autophagy. Using recombinant human asporin, we demonstrated that asporin stimulates an increase in LC3 and p62 in parallel to a decrease in COX IV and SMAD2 phosphorylation, indicating mitophagy induction and a decrease in TGFβ signaling. Based on our preliminary data and sequence comparison to a previously reported human peptide, we developed a rodent asporin peptide which is predicted to induce mitophagy while decreasing TGFβ signaling to prevent fibrosis. Studies are ongoing to establish the clinical relevance of asporin in animal models of ischemic injury.

Collagen promotes matrix vesicle-mediated mineralization by vascular smooth muscle cells

Vascular calcification (VC) is a hallmark of atherosclerotic plaques. Calcification of advanced plaques shares common features with endochondral ossification of long bones and appears to be protective. On the other hand, microcalcification of early plaques, which is poorly understood, is thought to be harmful. Tissue-nonspecific alkaline phosphatase (TNAP) and collagen are the two proteins necessary for physiological mineralization. Here, we demonstrate the presence of membrane-bound TNAP, detected by immunofluorescence, that seems to form clusters on the plasma membrane of vascular smooth muscle cells (VSMCs) cultured in mineralizing conditions. We observed that TNAP activity and mineralization were increased when VSMCs were cultured in the presence of ascorbic acid (AA) and β-glycerophosphate (β-GP). Increased TNAP activity was observed in whole cell lysates, total membrane fractions and, more particularly, in matrix vesicles (MVs). We have shown that TNAP-enriched MVs released from VSMCs subjected to collagenase contained more apatite-like mineral than the less TNAP-rich/TNAP-enriched vesicles isolated without collagenase treatment. These results suggest a role for collagen in promoting calcification induced by TNAP in atherosclerotic plaques.

Investigation of Relative Contribution of Intestinal and Hepatic UGT2B17 on Testosterone First‐Pass Metabolism

Background and ObjectiveOral testosterone show poor and unpredictable bioavailability due to high and variable first‐pass metabolism. We have demonstrated that testosterone glucuronidation is mediated by UGT2B17 (major) and UGT2B15 (minor) in human liver1. In this study, we compared 1) UGT2B17 protein abundance in microsomes and primary cells isolated from human intestine vs. liver, and 2) testosterone glucuronidation activity in primary human enterocytes vs. hepatocytes.MethodsHuman intestinal microsomes (HIMs) were isolated from different sections of intestine, i.e., duodenum (D), jejunum (J1–J3), ileum (I), and colon (C), from individual donors (n=7). Total membrane fractions were isolated from hepatocytes (n=5, Lonza) and enterocytes (n=16, IVAL). HIM, membrane fractions, and pooled liver and intestinal S9 fractions (Xenotech) were digested using optimized methods. Surrogate peptides of UGT2B17 and/or UGT2B15 were quantified by a validated LC‐MS/MS method. Testosterone glucuronidation activity was assessed in suspended hepatocytes and enterocytes as well as in S9 fractions at 5–50 μM concentrations at 37 °C. Reactions were quenched after 30–120 min, supernatants were analyzed via LC‐MS/MS, and data were normalized to total protein. Data comparisons were done using non‐parametric tests.ResultsUGT2B17 abundance was highly variable in both HIMs (&gt;9‐fold) and human liver microsomes (HLMs) (&gt;170‐fold), and was undetectable in 17% of HIMs and 45% of HLMs. When detected, UGT2B17 protein abundance (pmol/mg protein, mean ±SD) was 4.6‐fold higher (p&lt;0.001) in entire small intestine (8.9 ± 3.0) vs. liver (1.9 ± 2.6)1. Noticeably, we observed an increasing trend along the gastrointestinal (GI) segments (D, 7.2 ± 8.6; J1, 5.9 ± 4.7; J2, 7.2 ± 4.3; J3, 11.1 ± 7.2; I, 12.9 ± 10.5; and C, 19.4 ± 9.1). Mean UGT2B17 protein abundance in UGT2B17‐expressing enterocytes (0.47 ± 0.52) was higher compared to hepatocytes (0.22 ± 0.27). UGT2B15 protein abundance in hepatocytes was 3.0 ± 1.7 pmol/mg protein but undetectable in enterocytes. Similar trend was observed in testosterone glucuronidation activity (pmol/min/mg protein) in enterocytes vs. hepatocytes (32.3 ± 25.0 vs. 18.6 ± 10.3). Higher activity and/or expression of UGT2B17 were consistent in the pooled intestine vs. liver (S9 and microsomes).DiscussionUnavailability of oral testosterone formulation is an unmet need in the growing US market for testosterone replacement therapy. Overall higher UGT2B17 abundance in the GI compared to liver indicate a greater magnitude of testosterone first‐pass metabolism in the GI vs. liver, as reflected by the higher activity in enterocytes. Fractional contribution of UGT2B17 in testosterone first‐pass metabolism is predicted to be much greater in the GI vs. liver due to undetectable UGT2B15 in the GI, which postulates that variable testosterone bioavailability2 is likely primarily due to the intestinal UGT2B17. Consideration of differential UGT2B17 expression along GI tract is important in designing testosterone oral formulation for targeted absorption. Further studies are warranted to investigate physiological relevance of high UGT2B17 abundance in distal GI segments. Nevertheless, these observational data are crucial in constructing a PBPK model of oral testosterone and other UGT2B17 substrates.Support or Funding InformationR01 HD081299This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of placental nutrient transporters associated with intrauterine growth restriction and pre-eclampsia

BackgroundGestational disorders such as intrauterine growth restriction (IUGR) and pre-eclampsia (PE) are main causes of poor perinatal outcomes worldwide. Both diseases are related with impaired materno-fetal nutrient transfer, but the crucial transport mechanisms underlying IUGR and PE are not fully elucidated. In this study, we aimed to identify membrane transporters highly associated with transplacental nutrient deficiencies in IUGR/PE.ResultsIn silico analyses on the identification of differentially expressed nutrient transporters were conducted using seven eligible microarray datasets (from Gene Expression Omnibus), encompassing control and IUGR/PE placental samples. Thereby 46 out of 434 genes were identified as potentially interesting targets. They are involved in the fetal provision with amino acids, carbohydrates, lipids, vitamins and microelements. Targets of interest were clustered into a substrate-specific interaction network by using Search Tool for the Retrieval of Interacting Genes. The subsequent wet-lab validation was performed using quantitative RT-PCR on placentas from clinically well-characterized IUGR/PE patients (IUGR, n = 8; PE, n = 5; PE+IUGR, n = 10) and controls (term, n = 13; preterm, n = 7), followed by 2D-hierarchical heatmap generation. Statistical evaluation using Kruskal-Wallis tests was then applied to detect significantly different expression patterns, while scatter plot analysis indicated which transporters were predominantly influenced by IUGR or PE, or equally affected by both diseases. Identified by both methods, three overlapping targets, SLC7A7, SLC38A5 (amino acid transporters), and ABCA1 (cholesterol transporter), were further investigated at the protein level by western blotting. Protein analyses in total placental tissue lysates and membrane fractions isolated from disease and control placentas indicated an altered functional activity of those three nutrient transporters in IUGR/PE.ConclusionsCombining bioinformatic analysis, molecular biological experiments and mathematical diagramming, this study has demonstrated systematic alterations of nutrient transporter expressions in IUGR/PE. Among 46 initially targeted transporters, three significantly regulated genes were further investigated based on the severity and the disease specificity for IUGR and PE. Confirmed by mRNA and protein expression, the amino acid transporters SLC7A7 and SLC38A5 showed marked differences between controls and IUGR/PE and were regulated by both diseases. In contrast, ABCA1 may play an exclusive role in the development of PE.

Antigenic Properties of Iron Regulated Proteins in Acinetobacter baumannii: An In Silico Approach

Acinetobacter baumannii, an emerging nosocomial pathogen, causes multi-drug and pan-drug resistant infections. This phenomenon necessitates the development of new treatment strategies or vaccines against this pathogen. Iron acquisition systems are important factors for the virulence of pathogenic organisms. Antibodies against iron regulated outer membrane proteins (IROMPs) showed bactericidal and opsonizing activities against A. baumannii in vitro. Data obtained from proteomic studies could be valuable for vaccine design. Comparative proteomic analysis of total lysate and outer membrane fractions isolated from A. baumannii ATCC 19606 cells cultured under iron-rich and chelated conditions resulted in the identification of protein spots differentially produced. Need for bioinformatic tools is imperative for analysis of these data as well as to design novel proteins bearing desired properties. This study undertakes in silico identification of the most effective immunogenic target amongst these iron regulated proteins in A. baumannii which can be employed as a vaccine candidate. Here a screening was carried out on iron- regulated A. baumannii OMPs, based on hydrophilicity, flexibility, beta-turns, solubility and overall antigenic probability. 3D structure of the proteins was modeled. Linear and conformational B cell epitopes predicted and their densities were used for comparison of their immunogenicity. CarO was selected as an efficient immunogenic IROMP in A. baumannii which contributes to sidrophore mediated iron uptake.