High Molecular Mass Species Research Articles

Tyrosine Nitration Contributes to Nitric Oxide-Stimulated Degradation of CYP2B6.

Human cytochrome P450 (P450) CYP2B6 undergoes nitric oxide (NO)-dependent proteasomal degradation in response to the NO donor dipropylenetriamine NONOate (DPTA) and biologic NO in HeLa and HuH7 cell lines. CYP2B6 is also downregulated by NO in primary human hepatocytes. We hypothesized that NO or derivative reactive nitrogen species may generate adducts of tyrosine and/or cysteine residues, causing CYP2B6 downregulation, and selected Tyr and Cys residues for mutation based on predicted solvent accessibility. CYP2B6V5-Y317A, -Y380A, and -Y190A mutant proteins expressed in HuH7 cells were less sensitive than wild-type (WT) enzyme to degradation evoked by DPTA, suggesting that these tyrosines are targets for NO-dependent downregulation. The Y317A or Y380A mutants did not show increases in high molecular mass (HMM) species after treatment with DPTA or bortezomib + DPTA, in contrast to the WT enzyme. Carbon monoxide-releasing molecule 2 treatment caused rapid suppression of 2B6 enzyme activity, significant HMM species generation, and ubiquitination of CYP2B6 protein but did not stimulate CYP2B6 degradation. The CYP2B6 inhibitor 4-(4-chlorophenyl)imidazole blocked NO-dependent CYP2B6 degradation, suggesting that NO access to the active site is important. Molecular dynamics simulations predicted that tyrosine nitrations of CYP2B6 would cause significant destabilizing perturbations of secondary structure and remove correlated motions likely required for enzyme function. We propose that cumulative nitrations of Y190, Y317, and Y380 by reactive nitrogen species cause destabilization of CYP2B6, which may act synergistically with heme nitrosylation to target the enzyme for degradation. SIGNIFICANCE STATEMENT: This work provides novel insight into the mechanisms by which nitric oxide, which is produced in hepatocytes in response to inflammation, triggers the ubiquitin-dependent proteasomal degradation of the cytochrome P450 (P450) enzyme CYP2B6. Our data demonstrate that both nitration of specific tyrosine residues and interaction of nitric oxide (NO) with the P450 heme are necessary for NO to trigger ubiquitination and protein degradation.

Nitric Oxide Mediated Degradation of CYP2A6 via the Ubiquitin-Proteasome Pathway in Human Hepatoma Cells.

Several cytochrome P450 enzymes are known to be down-regulated by nitric oxide (NO). CYP2A6 is responsible for the metabolism of nicotine and several other xenobiotics, but its susceptibility to down-regulation by NO has not been reported. To address this question, we used Huh7 human hepatoma cell lines to express CYP2A6 with a C-terminal V5 tag (CYP2A6V5). NO donor treatment [dipropylenetriamine NONOate (DPTA)] down-regulated CYP2A6 protein to approximately 40% of control levels in 4 hours. An NO scavenging agent protected CYP2A6 from down-regulation by DPTA in a concentration-dependent manner, demonstrating that the down-regulation is NO-dependent. Experiments with the protein synthesis inhibitor cycloheximide showed that CYP2A6 protein down-regulation occurs posttranslationally. In the presence of proteasome inhibitors MG132 or bortezomib, NO-treated cells showed an accumulation of a high molecular mass signal, whereas autophagy inhibitors chloroquine and 3-methyladenine and the lysosomal and calpain inhibitor E64d had no effect. Immunoprecipitation of CYP2A6 followed by Western blotting with an antiubiquitin antibody showed that the high molecular mass species contain polyubiquitinated CYP2A6 protein. This suggests that NO led to the degradation of protein via the ubiquitin-proteasome pathway. The down-regulation by NO was blocked by the reversible CYP2A6 inhibitor pilocarpine but not by the suicide inhibitor methoxsalen, demonstrating that down-regulation requires NO access to the active site but does not require catalytic activity of the enzyme. These findings provide novel insights toward the regulation of CYP2A6 in a human cell line and can influence our understanding of CYP2A6-related drug metabolism. SIGNIFICANCE STATEMENT: This study demonstrates that the nicotine metabolizing enzyme CYP2A6 is down-regulated by nitric oxide, a molecule produced in large amounts in the context of inflammation and that is also inhaled from cigarette smoke. This occurs via ubiquitination and proteasomal degradation, and does not require catalytic activity of the enzyme. This work adds to the growing knowledge of the selective effect and mechanism of action of nitric oxide (NO) on cytochrome P450 enzymes and suggests a possible novel mode of interaction between nicotine and NO in cigarette smokers.

Structural Characterization of the Aggregates of Gemtuzumab Ozogamicin

The preparation of antibody drug conjugates (ADCs), particularly those containing hydrophobic payloads, may promote aggregate formation. The aggregate (high molecular mass species, HMMS) is an impurity that must be controlled in the final drug substance and drug product formulation to meet product safety and efficacy. Although there are numerous methods designed to mitigate protein aggregation, some of the reasons for the formation of aggregates in ADCs are unique and specific to the synthetic preparation and structure of each conjugate. Proper structural characterization and identification of causes of aggregation are key to improving the process for preparation of ADCs for minimizing aggregate formation. In this article, we have characterized aggregates generated during the preparation of a lysine conjugate, gemtuzumab ozogamicin, prepared from an IgG4 antibody (hP67.6) and a calicheamicin-based linker-payload. Using analytical and biophysical techniques, structural details of the aggregates formed in t...

Nitric oxide-regulated proteolysis of human CYP2B6 via the ubiquitin-proteasome system

We showed previously that rat cytochrome P450 CYP2B1 undergoes NO-dependent proteasomal degradation in response to inflammatory stimuli, and that the related human enzyme CYP2B6 is also down-regulated by NO in primary human hepatocytes. To investigate the mechanism of CYP2B6 down-regulation, we made several cell lines (HeLa and HuH7 cells) in which native CYP2B6 or CYP2B6 with a C-terminal V5 tag (CYP2B6V5) are expressed from a lentiviral vector with a cytomegalovirus promoter. Native CYP2B6 protein was rapidly down-regulated in HeLa cells within 3h of treatment with the NO donor (Z)−1-[2-(2-Aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate, while its mRNA level was not down-regulated. Treatment of the cells with the NO donor (Z)−1-[N-(3-aminopropyl)-N-(3-ammoniopropyl)amino]diazen-1-ium-1,2-diolate also resulted in rapid down-regulation of CYP2B6 activity, measured as the formation of 7-hydroxy-4-trifluoromethylcoumarin, as well as 2B6 protein in the CYP2B6 HeLa cell line. CYP2B6V5 was also down-regulated by NO donors in HuH7 cells. Down-regulation was observed in the presence of cycloheximide, demonstrating that this occurs via a post-translational mechanism. We generated a HeLa cell line expressing both CYP2B6V5 and human nitric oxide synthase 2 (NOS2), the latter under positive control by tetracycline. The cellular NO produced by doxycycline treatment also effectively down-regulated CYP2B6 protein, which was blocked by the co-treatment with the NOS2 competitive inhibitor L-NG-nitroarginine methyl ester (L-NAME). We next investigated the proteolytic enzymes responsible for NO-dependent CYP2B6 degradation. Neither calpain inhibitors (N-Acetyl-L-leucyl-L-leucyl-L-norleucinal, carbobenzoxy-valinyl-phenylalaninal), nor lysosomal protease inhibitors (3-methyladenine and chloroquine) inhibited the NO dependent CYP2B6V5 down-regulation. The proteasome inhibitors MG132 and bortezomib attenuated, but did not completely block the NO-induced down-regulation in the HuH7 cell line. However, when cells were co-treated with NO donor and proteasome inhibitors, high molecular mass species could be detected on native CYP2B6 as well as CYP2B6V5 Western blots. Further investigation demonstrated that CYP2B6 protein was polyubiquitinated and this was dramatically enhanced by co-treatment with NO donor and bortezomib. Taken together, our data demonstrate that CYP2B6 is down-regulated in an NO-dependent manner via ubiquitination and proteasomal degradation.

Accurate calibration of a molecular beam time-of-flight mass spectrometer for on-line analysis of high molecular weight species.

Time-of-Flight (TOF) Mass Spectrometry is a powerful analytical technique, provided that an accurate calibration by standard molecules in the same m/z range of the analytes is performed. Calibration in a very large m/z range is a difficult task, particularly in studies focusing on the detection of high molecular weight clusters of different molecules or high molecular weight species. External calibration is the most common procedure used for TOF mass spectrometric analysis in the gas phase and, generally, the only available standards are made up of mixtures of noble gases, covering a small mass range for calibration, up to m/z 136 (higher mass isotope of xenon). In this work, an accurate calibration of a Molecular Beam Time-of Flight Mass Spectrometer (MB-TOFMS) is presented, based on the use of water clusters up to m/z 3000. The advantages of calibrating a MB-TOFMS with water clusters for the detection of analytes with masses above those of the traditional calibrants such as noble gases were quantitatively shown by statistical calculations. A comparison of the water cluster and noble gases calibration procedures in attributing the masses to a test mixture extending up to m/z 800 is also reported. In the case of the analysis of combustion products, another important feature of water cluster calibration was shown, that is the possibility of using them as "internal standard" directly formed from the combustion water, under suitable experimental conditions. The water clusters calibration of a MB-TOFMS gives rise to a ten-fold reduction in error compared to the traditional calibration with noble gases. The consequent improvement in mass accuracy in the calibration of a MB-TOFMS has important implications in various fields where detection of high molecular mass species is required. In combustion products analysis, it is also possible to obtain a new calibration spectrum before the acquisition of each spectrum, only modifying some operative conditions. Copyright © 2016 John Wiley & Sons, Ltd.

Inhibiting Hexamer Disassembly of Human UDP-Glucose Dehydrogenase by Photoactivated Amino Acid Cross-Linking.

The enzyme UDP-glucose dehydrogenase (UGDH) catalyzes the reaction of UDP-glucose to UDP-glucuronate through two successive NAD(+)-dependent oxidation steps. Human UGDH apoprotein is purified as a mixture of dimeric and hexameric species. Addition of substrate and cofactor stabilizes the oligomeric state to primarily the hexameric form. To determine if the dynamic conformations of hUGDH are required for catalytic activity, we used site-specific unnatural amino acid incorporation to facilitate cross-linking of monomeric subunits into predominantly obligate oligomeric species. Optimal cross-linking was achieved by encoding p-benzoyl-l-phenylalanine at position 458, normally a glutamine located within the dimer-dimer interface, and exposing the enzyme to long wavelength ultraviolet (UV) radiation in the presence of substrate and cofactor. Hexameric complexes were purified by gel filtration chromatography and found to contain significant fractions of dimer and trimer (approximately 50%) along with another 10% higher-molecular mass species. The activity of the cross-linked enzyme was reduced by almost 60% relative to that of the un-cross-linked UGDH mutant, and UV exposure had no effect on the activity of the wild-type enzyme. These results support a model for catalysis in which the ability to dissociate the dimer-dimer interface is as important for maximal enzyme function as has been previously shown for the formation of the hexamer.

Membrane assisted recovery and purification of bio-based succinic acid for improved process sustainability

Three membrane-assisted technologies – electrodialysis, nanofiltration, and Donnan dialysis – were investigated with the objective of developing a green and sustainable integrated downstream process for the recovery of biologically produced succinic acid from carob pod-based fermentation broths. For electrodialysis, heterogeneous and homogenous anion-exchange membranes (including such with monovalent-anion-permselective transport properties) were tested with synthetic model solutions in order to select the most suitable membrane. For nanofiltration, six polymeric membranes were tested to filter the obtained electrodialysis concentrates, and according to the results obtained, three of them (NF270, NF-DK and NF-DL) were selected for further experiments. The performance of these membranes was compared in a diafiltration operation mode with model carboxylates solutions and with simulated fermentation broths, all showing rejections above 90% for succinate and reaching negative rejections for acetate and formate. However, reduced fluxes and fouling were observed in the experiment with simulated fermentation broth containing a carob pod extract. Therefore this medium was treated by electrodialysis, producing a concentrate rich in ionic species, including succinate, but leaving behind the non-ionic and high-molecular mass species. This concentrate was treated by nanofiltration, thus achieving an almost complete removal of accompanying formate and acetate species. Finally, Donnan dialysis was employed to replace the metallic cations in the succinate salts in the nanofiltration retentate by H+. The results showed approximately 76% of cations removal, which was traduced in the same percentage of obtained free succinic acid. Thus, the proposed integrated membrane-assisted process represents a promising new alternative to the existing downstream technologies for bio-based succinic acid recovery.

Development of a modular virus clearance package for anion exchange chromatography operated in weak partitioning mode.

Anion exchange chromatography (AEX) operated under weak partitioning mode has been proven to be a powerful polishing step as well as a robust viral clearance step in Pfizer's monoclonal antibody (mAb) platform purification process. A multivariate design of experiment (DoE) study was conducted to understand the impact of operating parameters and feedstream impurity levels on viral clearance by weak partitioning mode AEX. Bacteriophage was used initially as a surrogate for neutral and acidic isoelectric point mammalian viruses (e.g., retrovirus and parvovirus). Five different mAbs were used in the evaluation of process parameters such as load challenge (both product and impurities), load pH, load conductivity, and contact time (bed height and flow-rate). The operating ranges obtained from phage clearance studies and Pfizer's historical data were used to define an appropriate operating range for a subsequent clearance study with model retrovirus and parvovirus. Both phage and virus clearance evaluations included feedstreams containing different levels of impurities such as high molecular mass species (HMMS), host cell proteins (HCPs), and host cell DNA. For all the conditions tested, over 5 log10 of clearance for both retrovirus and parvovirus was achieved. The results demonstrated that weak partitioning mode AEX chromatography is a robust step for viral clearance and has the potential to be included as part of the modular viral clearance approach.

Molecular attributes of conjugate antigen influence function of antibodies induced by anti-nicotine vaccine in mice and non-human primates.

Anti-nicotine vaccines aim to prevent nicotine entering the brain, and thus reduce or eliminate the reward that drives nicotine addiction. Those tested in humans to date have failed to improve quit rates over placebo, possibly because antibody (Ab) responses were insufficient to sequester enough nicotine in the blood in the majority of subjects. We have previously shown in mice that the carrier, hapten and linker used in the nicotine conjugate antigen each influence the function (nicotine-binding capacity) of the Ab induced. Herein we have evaluated immunogenicity in mice of 27 lots of NIC7-CRM, a conjugate of 5-aminoethoxy-nicotine (Hapten 7) and a mutant nontoxic form of diphtheria toxin (CRM197), that differed in three antigen attributes, namely hapten load (number of haptens conjugated to each molecule of CRM197), degree of conjugate aggregation and presence of adducts (small molecules attached to CRM197 via a covalent bond during the conjugation process). A range of functional responses (reduced nicotine in the brain of immunized animals relative to non-immunized controls) were obtained with the different conjugates, which were adjuvanted with aluminum hydroxide and CpG TLR9 agonist. Trends for better functional responses in mice were obtained with conjugates having a hapten load of 11 to 18, a low level of high molecular mass species (HMMS) (i.e., not aggregated) and a low level of adducts and a more limited testing in cynomolgus monkeys confirmed these results. Thus hapten load, conjugate aggregation and presence of adducts are key antigen attributes that can influence Ab function induced by NIC7-CRM.

Characterization of biomass burning emissions from cooking fires, peat, crop residue, and other fuels with high-resolution proton-transfer-reaction time-of-flight mass spectrometry

Abstract. We deployed a high-resolution proton-transfer-reaction time-of-flight mass spectrometer (PTR-TOF-MS) to measure biomass-burning emissions from peat, crop residue, cooking fires, and many other fire types during the fourth Fire Lab at Missoula Experiment (FLAME-4) laboratory campaign. A combination of gas standard calibrations and composition sensitive, mass-dependent calibration curves was applied to quantify gas-phase non-methane organic compounds (NMOCs) observed in the complex mixture of fire emissions. We used several approaches to assign the best identities to most major "exact masses", including many high molecular mass species. Using these methods, approximately 80–96% of the total NMOC mass detected by the PTR-TOF-MS and Fourier transform infrared (FTIR) spectroscopy was positively or tentatively identified for major fuel types. We report data for many rarely measured or previously unmeasured emissions in several compound classes including aromatic hydrocarbons, phenolic compounds, and furans; many of these are suspected secondary organic aerosol precursors. A large set of new emission factors (EFs) for a range of globally significant biomass fuels is presented. Measurements show that oxygenated NMOCs accounted for the largest fraction of emissions of all compound classes. In a brief study of various traditional and advanced cooking methods, the EFs for these emissions groups were greatest for open three-stone cooking in comparison to their more advanced counterparts. Several little-studied nitrogen-containing organic compounds were detected from many fuel types, that together accounted for 0.1–8.7% of the fuel nitrogen, and some may play a role in new particle formation.

Agitation-induced aggregation and subvisible particulate formation in model proteins

The kinetics of agitation-induced subvisible particle formation was investigated for a few model proteins – human serum albumin (HSA), hen egg white lysozyme (HEWL), and a monoclonal antibody (IgG2). Experiments were carried out for the first time under relatively low protein concentration and low agitation speed to investigate the details of subvisible particle formation at the initial phase of aggregation (<2%) process. Upon agitation, both soluble higher molecular mass species (HMMS) and subvisible particles (SbVPs) formed at different rates, and via different mechanisms. Agitation enhanced exposure of hydrophobic sites in HSA but did not cause detectable structural changes in HEWL and IgG2. SbVPs from HSA partially dissociates in a neutral pH buffer (SEC mobile phase) but does not upon dilution in the same formulation buffer. Opposite results were obtained for SbVPs from IgG2 and HEWL. Neither the relative hydrophobic surface area nor the Tm of the model proteins seems to be an indicator of tendency for agitation-mediated SbVP formation. Taken together, our data suggests that agitation-induced SbVP formation can occur through different mechanisms and can vary, depending on the protein and solution conditions.

Aluminium and Other Metals May Pose a Risk to Children with Autism Spectrum Disorder: Biochemical and Behavioural Impairments

Recent studies have shown that some toxic metals have been associated with neurological diseases. The binding and transport of these metals in the blood may occur by species of High Molecular Mass (HMM) and Low Molecular Weight Species (LMM) of proteins. These main species are known to be responsible for aluminum transporting is the case of transferrin and citrate. It seems that autistic children may have a genetic predisposition to accumulate large amounts of metals as it is has been proposed. This study seeks to understand the toxic mechanism of selected metals in autistic children and its correlation with the psycho-metabolic implications of the syndrome. Preliminary results have indicated that some metals such as chromium, arsenic and particularly aluminum were elevated in the blood of an autistic child, as compared to reference values of a normal child. A case-control study is under investigation. Furthermore, the consequences of the disease, as such the difficulties in socialization and language skill disabilities may also be related to the burden of toxic metals in general, particularly aluminum.

SEPARATION OF GALACTOGLUCOMANNANS, LIGNIN, AND LIGNIN-CARBOHYDRATE COMPLEXES FROM HOT-WATER-EXTRACTED NORWAY SPRUCE BY CROSS-FLOW FILTRATION AND ADSORPTION CHROMATOGRAPHY

A simple method to simultaneously recover polymeric carbohydrates, mainly galactoglucomannans (GGM), lignin, and lignin-carbohydrate complex (LCC) from hot-water-extracted Norway spruce wood is presented. The isolation method consists of cross-flow filtration, where high and low molecular mass species are removed, followed by fixed-bed adsorption on a hydrophobic polymeric resin (XAD-16) to remove lignins and lignans. In the second step of fixed-bed adsorption, a phenylic reversed-phase analytical chromatography column, where mass transport resistance is minimized and a very high selectivity towards aromatic compounds have been observed, was used to separate LCC from GGM. The isolated LCC fraction contained about 10% aromatics, whereas the upgraded GGM fraction contained about 1.5% aromatics and the lignin fraction contained about 56% aromatics. Polymeric xylan was accumulated in the GGM fraction, while mannose was the dominant sugar found in the LCC fraction. As products, approximately 7% was recovered in the lignin fraction in the first adsorptive step, 5% was recovered as LCC, and 88% as upgraded hemicelluloses.

Study on Lignin–Glyoxal Reaction by MALDI-TOF and CP-MAS 13C-NMR

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry was used to elucidate lignin–glyoxal reactions. The reaction of glyoxal with a low molecular weight lignin involves a number of different reactions; in particular lignin depolymerization and recombination through condensation reactions with glyoxal to form glyoxalene bridges linking both lignin units and mainly fractions of lignin units derived from degradation during depolymerization. The reactive hydroxyglyoxalated lignin is the one species still available for co-reaction for use in wood adhesives. The reactions described increase the viscosity of the lignin as the reaction proceeds towards higher molecular mass species and the condensed species grow in relative proportions. NMR analysis confirmed the existence of functional groups pertaining to structures that are formed from glyoxalation of the lignin observed by some results obtained by MALDI-TOF. MALDI-TOF mass spectrometry appears to be a suitable method for examining lignin–glyoxal reactions.

Isolation and Compositional Analysis of a CP12-Associated Complex of Calvin Cycle Enzymes from Nicotiana tabacum

Two Calvin Cycle enzymes, NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a multiprotein complex with CP12, a small intrinsically-unstructured protein. Under oxidizing conditions, association with CP12 confers redox-sensitivity to the otherwise redox-insensitive A isoform of GAPDH (GapA) and provides an additional level of down-regulation to the redox-regulated PRK. To determine if CP12-mediated regulation is specific for GAPDH and PRK in vivo, a high molecular weight complex containing CP12 was isolated from tobacco chloroplasts and leaves and its protein composition was characterized. Gel electrophoresis and immunoblot analyses after separation of stromal proteins by size fractionation verified that the GAPDH (both isoforms) and PRK co-migrated with CP12 in dark- but not light-adapted chloroplasts. Nano-liquid-chromatography-mass-spectrometry of the isolated complex identified only CP12, GAPDH and PRK. Since nearly all of the CP12 from darkened chloroplasts migrates with GADPH and PRK as a high molecular mass species, these data indicate that the tight association of tobacco CP12 with GAPDH and PRK is specific and involves no other Calvin Cycle enzymes.

Human T-lymphotropic Virus Type 1 p30 Interacts with REGγ and Modulates ATM (Ataxia Telangiectasia Mutated) to Promote Cell Survival

Human T-lymphotropic virus type 1 (HTLV-1) is a causative agent of adult T cell leukemia/lymphoma and a variety of inflammatory disorders. HTLV-1 encodes a nuclear localizing protein, p30, that selectively alters viral and cellular gene expression, activates G(2)-M cell cycle checkpoints, and is essential for viral spread. Here, we used immunoprecipitation and affinity pulldown of ectopically expressed p30 coupled with mass spectrometry to identify cellular binding partners of p30. Our data indicate that p30 specifically binds to cellular ATM (ataxia telangiectasia mutated) and REGγ (a nuclear 20 S proteasome activator). Under conditions of genotoxic stress, p30 expression was associated with reduced levels of ATM and increased cell survival. Knockdown or overexpression of REGγ paralleled p30 expression, suggesting an unexpected enhancement of p30 expression in the presence of REGγ. Finally, size exclusion chromatography revealed the presence of p30 in a high molecular mass complex along with ATM and REGγ. On the basis of our findings, we propose that HTLV-1 p30 interacts with ATM and REGγ to increase viral spread by facilitating cell survival.

Rapid fractionation of Al in human serum by the use of HiTrap desalting size exclusion column with ICP-MS detection

To understand the pathways of Al transport in human body it is necessary to identify and quantify Al species present in human serum. It has been demonstrated that about 90% of Al in human serum corresponds to high molecular mass species (HMM-Al) and that Al is bound exclusively to transferrin (Tf). The remaining Al exists in low molecular mass species (LMM-Al) bound predominantly to Al-citrate. In studies of the efficiencies of the chelation therapies and to perform kinetic studies of Al binding to Tf, rapid and reliable analytical procedures are required. Therefore, a new analytical procedure for the efficient, reliable and fast separation of proteins from LMM compounds in serum was developed by the use of a HiTrap desalting size exclusion column. Unspiked and spiked human serum that was left for six hours to reach approximately thermodynamic equilibrium were studied. The separation of HMM-Al from LMM-Al species was accomplished with tris-HCl buffer (pH 7.4) in 10 min. Al elution profile was followed by ICP-MS detection. In the first 5 min HMM-Al compounds were eluted, followed by the elution of LMM-Al species from 5 to 10 min. Results demonstrated that about 94% of Al was present in the HMM (protein) fraction. To prove the reliability of the fractionation procedure developed, the protein peak and LMM separated species were collected in 5 mL fractions, followed by the previously developed speciation procedures. Speciation of the protein peak by CIM® DEAE-ICP-MS verified that about 98% of Al was bound to Tf. In the LMM serum fraction Al-citrate was separated on the FPLC column coupled to ICP-MS. Speciation data confirmed the reliability of the developed fast fractionation procedure. It represents a promising tool for investigations of kinetics of Al binding to Tf and may also be applied in studies of the efficiencies of the chelation therapies in patients overloaded with Al.

Heteromeric Interactions Required for Abundance and Subcellular Localization of Human CDC50 Proteins and Class 1 P4-ATPases

Members of the P(4) family of P-type ATPases (P(4)-ATPases) are believed to function as phospholipid flippases in complex with CDC50 proteins. Mutations in the human class 1 P(4)-ATPase gene ATP8B1 cause a severe syndrome characterized by impaired bile flow (intrahepatic cholestasis), often leading to end-stage liver failure in childhood. In this study, we determined the specificity of human class 1 P(4)-ATPase interactions with CDC50 proteins and the functional consequences of these interactions on protein abundance and localization of both protein classes. ATP8B1 and ATP8B2 co-immunoprecipitated with CDC50A and CDC50B, whereas ATP8B4, ATP8A1, and ATP8A2 associated only with CDC50A. ATP8B1 shifted from the endoplasmic reticulum (ER) to the plasma membrane upon coexpression of CDC50A or CDC50B. ATP8A1 and ATP8A2 translocated from the ER to the Golgi complex and plasma membrane upon coexpression of CDC50A, but not CDC50B. ATP8B2 and ATP8B4 already displayed partial plasma membrane localization in the absence of CDC50 coexpression but displayed a large increase in plasma membrane abundance upon coexpression of CDC50A. ATP8B3 did not bind CDC50A and CDC50B and was invariably present in the ER. Our data show that interactions between CDC50 proteins and class 1 P(4)-ATPases are essential for ER exit and stability of both subunits. Furthermore, the subcellular localization of the complex is determined by the P(4)-ATPase, not the CDC50 protein. The interactions of CDC50A and CDC50B with multiple members of the human P(4)-ATPase family suggest that these proteins perform broader functions in human physiology than thus far assumed.

Core glycan in the yeast multicopper ferroxidase, Fet3p: A case study of N‐linked glycosylation, protein maturation, and stability

Glycosylation is essential to the maintenance of protein quality in the vesicular protein trafficking pathway in eukaryotic cells. Using the yeast multicopper oxidase, Fet3p, the hypothesis is tested that core glycosylation suppresses Fet3p nascent chain aggregation during synthesis into the endoplasmic reticulum (ER). Fet3p has 11 crystallographically mapped N-linked core glycan units. Assembly of four of these units is specifically required for localization of Fet3p to the plasma membrane (PM). Fet3 protein lacking any one of these glycan units is found in an intracellular high-molecular mass species resolvable by blue native gel electrophoresis. Individually, the remaining glycan moieties are not required for ER exit; however, serial deletion of these by N → A substitution correlates with these desglycan species failure to exit the ER. Desglycan Fet3 proteins that localize to the PM are wild type in function indicating that the missing carbohydrate is not required for native structure and biologic activity. This native function includes the interaction with the iron permease, Ftr1p, and wild type high-affinity iron uptake activity. The four essential sequons are found within relatively nonpolar regions located in surface recesses and are strongly conserved among fungal Fet3 proteins. The remaining N-linked sites are found in more surface exposed, less nonpolar environments, and their conservation is weak or absent. The data indicate that in Fet3p the N-linked glycan has little effect on the enzyme's molecular activity but is critical to its cellular activity by maximizing the protein's exit from the ER and assembly into a functional iron uptake complex.

Tumor-Conditioned Macrophages Secrete Migration-Stimulating Factor: A New Marker for M2-Polarization, Influencing Tumor Cell Motility

Tumor-associated macrophages (TAMs) are key orchestrators of the tumor microenvironment directly affecting neoplastic cell growth, neoangiogenesis, and extracellular matrix remodeling. In turn, the tumor milieu strongly influences maturation of TAMs and shapes several of their features. To address the early macrophage (M) differentiation phase in a malignant context, we mimicked a tumor microenvironment by in vitro coculturing human blood monocytes with conditioned media from different cancer cell lines. Only 2 out of 16 tumor cell lines induced M differentiation due to secreted M-CSF isoforms, including high molecular mass species. A global gene profiling of tumor-conditioned M was performed. Comparison with other datasets (polarized M1-M, M2-M, and TAMs isolated from human tumors) highlighted the upregulation of several genes also shared by TAM and M2-polarized M. The most expressed genes were selenoprotein 1, osteoactivin, osteopontin, and, interestingly, migration-stimulating factor (MSF), a poorly studied oncofoetal isoform of fibronectin. MSF (present in fetal/cancer epithelial and stromal cells but not in healthy tissues) was never identified in M. MSF production was confirmed by immunohistochemistry in human TAMs. MSF was induced by M-CSF, IL-4, and TGFbeta but not by proinflammatory stimuli. RNA and protein analysis clearly demonstrated that it is specifically associated with the M2 polarization of M. Tumor-conditioned M-derived MSFs strongly stimulated tumor cell migration, thus contributing to the motile phenotype of neoplastic cells. In conclusion, MSF is a new molecule associated with the M2 polarization of M and expressed by TAMs. Its biological function may contribute to M-mediated promotion of cancer cell invasion and metastasis.