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Cholesterol synthesis is a tightly regulated process, both transcriptionally and post-translationally. Transcriptional control of cholesterol synthesis is relatively well-understood. However, of the ∼20 enzymes in cholesterol biosynthesis, post-translational regulation has only been examined for a small number. Three of the four sterol reductases in cholesterol production, 7-dehydrocholesterol reductase (DHCR7), 14-dehydrocholesterol reductase (DHCR14), and lamin-B receptor (LBR), share evolutionary ties with a high level of sequence homology and predicted structural homology. DHCR14 and LBR uniquely share the same Δ-14 reductase activity in cholesterol biosynthesis, yet little is known about their post-translational regulation. We have previously identified specific modes of post-translational control of DHCR7, but it is unknown whether these regulatory mechanisms are shared by DHCR14 and LBR. Using CHO-7 cells stably expressing epitope-tagged DHCR14 or LBR, we investigated the post-translational regulation of these enzymes. We found that DHCR14 and LBR undergo differential post-translational regulation, with DHCR14 being rapidly turned over, triggered by cholesterol and other sterol intermediates, whereas LBR remained stable. DHCR14 is degraded via the ubiquitin-proteasome system, and we identified several DHCR14 and DHCR7 putative interaction partners, including a number of E3 ligases that modulate DHCR14 levels. Interestingly, we found that gene expression across an array of human tissues showed a negative relationship between the C14-sterol reductases; one enzyme or the other tends to be predominantly expressed in each tissue. Overall, our findings indicate that whereas LBR tends to be the constitutively active C14-sterol reductase, DHCR14 levels are tunable, responding to the local cellular demands for cholesterol.

Crosstalk exists when two or more post-translational modifications, nearby in sequence or 3D space, affect each other or a protein's interactions. Saccharomyces cerevisiae protein Npl3p has six repeats of sequence SRGG, in a disordered domain, which can carry arginine methylation and serine phosphorylation. Crosstalk of the modifications controls Npl3p interactions with nuclear import, export, and other proteins. Here, we asked whether repeated SRGG motifs existed in other S. cerevisiae proteins and whether they serve a related function. Two other proteins had multiple SRGG motifs: Nop1p (fibrillarin) and Gar1p, both nucleolar proteins, which had nine and four motifs, respectively. For Nop1p, we first showed it to be extensively methylated in vivo. We then showed that the Nop1p SRGG motif is subjected to methylation by Hmt1p, phosphorylation by Sky1p, and Glc7p dephosphorylation and that there is crosstalk whereby phosphorylation blocks methylation. This is consistent with our recent motif analysis of Hmt1p, which revealed a negative specificity for acidic residues at −1 and −2 positions. On knockout of HMT1, Nop1p-GFP localization was not typically nucleolar. Conditional two-hybrid analysis, of Nop1p with C/D box small ribonuclear proteins Nop56p and Nop58p, suggested this may be associated with decreased protein-protein interactions on loss of arginine methylation. The effect of SRGG phosphorylation on the interactions of Nop1p remains unknown yet was predicted to cause a structural disorder-to-order transition in the Nop1p N-terminal domain. The SRGG motif is one of very few examples of modification crosstalk that has related functions in multiple proteins from the same species.

Systemic glutathione deficiency, inflammation, and oxidative stress are hallmarks of cystic fibrosis (CF), an inherited disease that causes persistent lung infections and severe damage to the respiratory system and many of the body organs. Improvements to current antioxidant therapeutic strategies are needed. The dietary supplement, &gamma, glutamylcysteine (GGC), which is the immediate precursor to glutathione, rapidly boosts cellular glutathione levels following a single dose in healthy individuals. Efficacy of GGC against oxidative stress induced by Pseudomonas aeruginosa, which is a common and chronic pathogen infecting lungs of CF patients, remains unassessed. Primary mucocilliary differentiated airway (bronchial and/or nasal) epithelial cells were created from four individuals with CF. Airway oxidative stress and inflammation was induced by P. aeruginosa lipopolysaccharide (LPS). Parameters including global proteomics alterations, cell redox state (glutathione, oxidative stress), pro-inflammatory mediators (IL-8, IDO-1), and cellular health (membrane integrity, stress granule formation, cell metabolic viability) were assayed under six experimental conditions: (1) Mock, (2) LPS-challenged (3) therapeutic, (4) prophylactic (5) therapeutic and prophylactic and (6) GGC alone. Proteomic analysis identified perturbation of several pathways related to cellular respiration and stress responses upon LPS challenge. Most of these were resolved when cells were treated with GGC. While GGC did not resolve LPS-induced IL-8 and IDO-1 activity, it effectively attenuated LPS-induced oxidative stress and stress granule formation, while significantly increasing total intracellular glutathione levels, metabolic viability and improving epithelial cell barrier integrity. Both therapeutic and prophylactic treatments were successful. Together, these findings indicate that GGC has therapeutic potential for treatment and prevention of oxidative stress-related damage to airways in cystic fibrosis.

IntroductionGlutathione deficiency and chronic bacterial inflammation exacerbates the oxidative stress damage to airways in cystic fibrosis. Improvements to current antioxidant therapeutic strategies are needed. Dietary supplement, γ-glutamylcysteine (GGC), the immediate precursor to glutathione, rapidly boosts cellular glutathione levels following a single dose in healthy individuals. Efficacy of GGC against Pseudomonas aeruginosa derived lipopolysaccharide (LPS), a prominent factor in mediating both bacterial virulence and host responses, in CF remains unassessed.MethodsPrimary F508del/F508del mucociliary differentiated bronchial and nasal epithelial cells were created to model LPS-induced oxidative stress and inflammation of CF. The proteomic signature of GGC treated cells was resolved by qLC-MS/MS. Parameters including cell redox state (glutathione, ROS), anti-inflammatory mediators (IL-8, IDO-1) and cellular health (membrane integrity, stress granule formation and cell viability) were assayed.ResultsProteomic analysis identified perturbation of several pathways related to cellular respiration and stress responses upon LPS challenge. Most of these were resolved when cells were treated with GGC. While GGC did not resolve LPS-induced IL-8 and IDO-1 activity, it effectively attenuated LPS-induced ROS and stress granule formation, while significantly increasing intracellular glutathione levels and improving epithelial cell barrier integrity. Moreover, we compared the effect of GGC with thiols NAC and glutathione on cell viability. GGC was the only thiol that increased cell viability; protecting cells against LPS induced cell death. Both therapeutic and prophylactic treatments were successful.ConclusionTogether, these findings indicate that GGC has therapeutic potential for treatment and prevention of oxidative stress related damage to airways in Cystic Fibrosis.

Hmt1p is the predominant arginine methyltransferase in Saccharomyces cerevisiae. Its substrate proteins are involved in transcription, transcriptional regulation, nucleocytoplasmic transport and RNA splicing. Hmt1p-catalyzed methylation can also modulate protein-protein interactions. Hmt1p is conserved from unicellular eukaryotes through to mammals where its ortholog, PRMT1, is lethal upon knockout. In yeast, however, the effect of knockout on the transcriptome and proteome has not been described. Transcriptome analysis revealed downregulation of phosphate-responsive genes in hmt1Δ, including acid phosphatases PHO5, PHO11, and PHO12, phosphate transporters PHO84 and PHO89 and the vacuolar transporter chaperone VTC3. Analysis of the hmt1Δ proteome revealed decreased abundance of phosphate-associated proteins including phosphate transporter Pho84p, vacuolar alkaline phosphatase Pho8p, acid phosphatase Pho3p and subunits of the vacuolar transporter chaperone complex Vtc1p, Vtc3p and Vtc4p. Consistent with this, phosphate homeostasis was dysregulated in hmt1Δ cells, showing decreased extracellular phosphatase levels and decreased total Pi in phosphate-depleted medium. In vitro, we showed that transcription factor Pho4p can be methylated at Arg-241, which could explain phosphate dysregulation in hmt1Δ if interplay exists with phosphorylation at Ser-242 or Ser-243, or if Arg-241 methylation affects the capacity of Pho4p to homodimerize or interact with Pho2p. However, the Arg-241 methylation site was not validated in vivo and the localization of a Pho4p-GFP fusion in hmt1Δ was not different from wild type. To our knowledge, this is the first study to reveal an association between Hmt1p and phosphate homeostasis and one which suggests a regulatory link between S-adenosyl methionine and intracellular phosphate.

This study investigated the enzyme-substrate interaction between Saccharomyces cerevisiae arginine methyltransferase Hmt1p and nucleolar protein Npl3p, using chemical cross linking/mass spectrometry (XL/MS). We show that XL/MS can capture transient interprotein interactions that occur during the process of methylation, involving a disordered region in Npl3p with tandem SRGG repeats, and we confirm that Hmt1p and Npl3p exist as homomultimers. Additionally, the study investigated the interdependencies between variables of an XL/MS experiment that lead to the identification of identical or different cross-linked peptides. We report that there are substantial benefits, in terms of biologically relevant cross-links identified, that result from the use of two mass-spectrometry-cleavable cross-linkers [disuccinimido sulfoxide (DSSO) and disuccinimido dibutyric urea (DSBU)], two fragmentation approaches [collision-induced dissociation and electron-transfer dissociation (CID+ETD)] and stepped high-energy collision dissociation (HCD)], and two programs (MeroX and XlinkX). We also show that there are specific combinations of XL/MS methods that are more successful than others for the two proteins investigated here; these are explored in detail in the text. Data are available via ProteomeXchange with identifier PXD008348.

The Family 16 methyltransferases are a group of eukaryotic nonhistone protein methyltransferases. Sixteen of these have recently been described in yeast and human, but little is known about their sequence and structural features. Here we investigate one of these methyltransferases, Saccharomyces cerevisiae elongation factor methyltransferase 2 (Efm2), by site‐directed mutagenesis and truncation. We show that an active site‐associated tryptophan, invariant in Family 16 methyltransferases and at position 222 in Efm2, is important for methyltransferase activity. A second highly conserved tryptophan, at position 318 in Efm2, is likely involved in S‐adenosyl methionine binding but is of lesser consequence for catalysis. By truncation analysis, we show that the N‐terminal 50–200 amino acids of Efm2 are critical for its methyltransferase activity. As N‐terminal regions are variable among Family 16 methyltransferases, this suggests a possible role in determining substrate specificity. This is consistent with recently solved structures that show the core of Family 16 methyltransferases to be near‐identical but the N termini to be structurally quite different. Finally, we show that Efm2 can exist as an oligomer but that its N terminus is not necessary for oligomerisation to occur.

Squalene monooxygenase (SM) is a rate-limiting enzyme in cholesterol synthesis. The region comprising the first 100 amino acids, termed SM N100, represents the shortest cholesterol-responsive degron and enables SM to sense excess cholesterol in the endoplasmic reticulum (ER) membrane. Cholesterol accelerates the ubiquitination of SM by membrane-associated ring-CH type finger 6 (MARCH6), a key E3 ubiquitin ligase involved in ER-associated degradation. However, the ubiquitination site required for cholesterol regulation of SM N100 is unknown. Here, we used SM N100 fused to GFP as a model degron to recapitulate cholesterol-mediated SM degradation and show that neither SM lysine residues nor the N terminus impart instability. Instead, we discovered four serines (Ser-59, Ser-61, Ser-83, and Ser-87) that are critical for cholesterol-accelerated degradation, with MS analysis confirming Ser-83 as a ubiquitination site. Notably, these two clusters of closely spaced serine residues are located in disordered domains flanking a 12-amino acid-long amphipathic helix (residues Gln-62-Leu-73) that together confer cholesterol responsiveness. In summary, our findings reveal the degron architecture of SM N100, introducing the role of non-canonical ubiquitination sites and deepening our molecular understanding of how SM is degraded in response to cholesterol.

High-resolution MS/MS spectra of peptides can be deisotoped to identify monoisotopic masses of peptide fragments. The use of such masses should improve protein identification rates. However, deisotoping is not universally used and its benefits have not been fully explored. Here, MS2-Deisotoper, a tool for use prior to database search, is used to identify monoisotopic peaks in centroided MS/MS spectra. MS2-Deisotoper works by comparing the mass and relative intensity of each peptide fragment peak to every other peak of greater mass, and by applying a set of rules concerning mass and intensity differences. After comprehensive parameter optimization, it is shown that MS2-Deisotoper can improve the number of peptide spectrum matches (PSMs) identified by up to 8.2% and proteins by up to 2.8%. It is effective with SILAC and non-SILAC MS/MS data. The identification of unique peptide sequences is also improved, increasing the number of human proteoforms by 3.7%. Detailed investigation of results shows that deisotoping increases Mascot ion scores, improves FDR estimation for PSMs, and leads to greater protein sequence coverage. At a peptide level, it is found that the efficacy of deisotoping is affected by peptide mass and charge. MS2-Deisotoper can be used via a user interface or as a command-line tool.

© The Royal Society of Chemistry. Polyelectrolyte-protein complexes are widely used to deliver therapeutic proteins. Here, we present a method for imaging the release of drugs from polyion complex (PIC) micelles in 3D tumour spheroids using light-sheet microscopy. A negatively charged block copolymer was condensed with a positively charged model drug, hen egg white lysozyme (HEWL) by electrostatic interaction. We were able to observe the distribution of polymer and protein within the entire tumour spheroid, showing that the protein was released from the polyelectrolyte complex upon cell internalization at the peripheral cell layer of the spheroid.

Protein methylation is one of the major post-translational modifications (PTMs) in the cell. In Saccharomyces cerevisiae, over 20 protein methyltransferases (MTases) and their respective substrates have been identified. However, the way in which these MTases are modified and potentially subject to regulation remains poorly understood. Here, we investigated six overexpressed S. cerevisiae protein MTases (Rkm1, Rkm4, Efm4, Efm7, Set5 and Hmt1) to identify PTMs of potential functional relevance. We identified 48 PTM sites across the six MTases, including phosphorylation, acetylation and methylation. Forty-two sites are novel. We contextualized the PTM sites in structural models of the MTases and revealed that many fell in catalytic pockets or enzyme–substrate interfaces. These may regulate MTase activity. Finally, we compared PTMs on Hmt1 with those on its human homologs PRMT1, PRMT3, CARM1, PRMT6 and PRMT8. This revealed that several PTMs are conserved from yeast to human, whereas others are only found in Hmt1. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD006767.

Post-translational lysine methylation is well established as a regulator of histone activity; however, it is emerging that these modifications are also likely to play extensive roles outside of the histone code. Here we obtain new insights into non-histone lysine methylation and protein lysine methyltransferase (PKMT) activity by elucidating absolute stoichiometries of lysine methylation, using mass spectrometry and absolute quantification (AQUA), in wild-type and 5 PKMT gene deletion strains of Saccharomyces cerevisiae. By analyzing 8 sites of methylation in 3 non-histone proteins, elongation factor 1-α (EF1α), elongation factor 2 (EF2), and 60S ribosomal protein L42-A/B (Rpl42ab), we find that production of preferred methylation states on individual lysine residues is commonplace and likely occurs through processive PKMT activity, Class I PKMTs can be associated with processive methylation, lysine residues are selectively methylated by specific PKMTs, and lysine methylation exists over a broad range of stoichiometries. Together these findings suggest that specific sites and forms of lysine methylation may play specialized roles in the regulation of non-histone protein activity. We also uncover new relationships between two proteins previously characterized as PKMTs, SEE1 and EFM1, in EF1α methylation and show that past characterizations of EFM1 as having direct PKMT activity may require reinterpretation.

Protein-protein interactions can be modulated by the methylation of arginine residues. As a means of testing this, we recently described a conditional two-hybrid system, based on the bacterial adenylate cyclase (BACTH) system. Here, we have used this conditional two-hybrid system to explore the effect of arginine methylation in modulating protein-protein interactions in a subset of the Saccharomyces cerevisiae arginine methylproteome network. Interactions between the yeast hub protein Npl3 and yeast proteins Air2, Ded1, Gbp2, Snp1, and Yra1 were first validated in the absence of methylation. The major yeast arginine methyltransferase Hmt1 was subsequently included in the conditional two-hybrid assay, initially to determine the degree of methylation that occurs. Proteins Snp1 and Yra1 were confirmed as Hmt1 substrates, with five and two novel arginine methylation sites mapped by ETD LC-MS/MS on these proteins, respectively. Proteins Ded1 and Gbp2, previously predicted but not confirmed as substrates of Hmt1, were also found to be methylated with five and seven sites mapped respectively. Air2 was found to be a novel substrate of Hmt1 with two sites mapped. Finally, we investigated the interactions of Npl3 with the five interaction partners in the presence of active Hmt1 and in the presence of Hmt1 with a G68R inactivation mutation. We found that the interaction between Npl3 and Air2, and Npl3 and Ded1, were significantly increased in the presence of active Hmt1; the interaction of Npl3 and Snp1 showed a similar degree of increase in interaction but this was not statistically significant. The interactions of Npl3 and Gbp2, along with Npl3 and Yra1, were not significantly increased or decreased by methylation. We conclude that methylarginine may be a widespread means by which the interactions of proteins are modulated.

Arginine methylation is a post-translational modification that has been implicated in a plethora of cellular processes. In the present manuscript, using two antimethylarginine antibodies and combinatorial deletion mutants of arginine methyltransferases, we found evidence of widespread arginine methylation in the Saccharomyces cerevisiae proteome. Immunoprecipitation was used for enrichment of methylarginine-containing proteins, which were identified via tandem mass spectrometry. From this, we identified a total of 90 proteins, of which 5 were previously known to be methylated. The proteins identified were involved in known methylarginine-associated biological functions such as RNA processing, nuclear transport, carbohydrate metabolic process, GMP biosynthetic process and protein folding. Through in vivo methylation by the incorporation of [3H]-methyl groups, we validated the methylation of 7 proteins (Ded1, Imd4, Lhp1, Nop1, Cdc11, Gus1, Pob3). By LC-MS/MS, we then confirmed a total of 15 novel methylarginine sites on 5 proteins (Ded1, Lhp1, Nop1, Pab1, and Ugp1). By examination of methylation on proteins from the triple knockout of methyltransferases Hmt1, Hsl7, Rmt2, we present evidence for the existence of additional unidentified arginine methyltransferases in the Saccharomyces cerevisiae proteome.

When localizing protein post-translational modifications (PTMs) using liquid-chromatography (LC)-tandem mass spectrometry (MS/MS), existing implementations are limited by inefficient selection of PTM-carrying peptides for MS/MS, particularly when PTM site occupancy is sub-stoichiometric. The present contribution describes a method by which peptides carrying specific PTMs of interest—in this study, methylarginines—may be selectively targeted for MS/MS: peptide features are extracted from high mass accuracy single-stage MS data, searched against theoretical PTM-carrying peptide masses, and matching features are subjected to targeted data acquisition LC-MS/MS. Using trypsin digested Saccharomyces cerevisiae Npl3, in which evidence is presented for 18 methylarginine sites—17 of which fall within a glycine-arginine-rich (GAR) domain spanning

For many decades, the capacity for mass spectrometry (MS) to analyse matterin all of its forms was shackled by an inability to ionise large molecules into the gas phase. It was only in the late 1980s, when the capabilities of the 'soft' ionisation protocols of matrix-assisted laser desorption/ ionisation (MALDI) and electrospray ionisation (ESI) became apparent, that these shackles were effectively removed. The emergence of soft ionisation methodologies acted as a driving force for significant advances in MS instrumentation; as a result, MS now represents one of the most potent means by which insights into the structural details of synthetic polymer samples can be obtained.However, despiteMSbeing acknowledged to be a mainstream analytical tool in the field of polymer chemistry, the number of MS-based studies into polymer systems remains significantly limited whencompared to other analytical methods, and it would thus appear that MS is still being severely underutilised. In this Trend article, important fundamental concepts of soft ionisation MS are presented in a tutorial style fashion within the context of the analysis of polymer systems, and the remarkable breadth, power andversatility of these techniques is illustrated using selected examples. We suggest that MS-based analyses represent one of the most promising areas of research in polymer science, and that the polymer chemistry community would bewell servedby utilising this analytical discipline in a more intensive manner. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Poly(acrylic acid) (polyAA), poly(N-isopropylacrylamide) (polyNIPAAm) and poly(diisopropyl vinyl phosphonate) (polyDISP) samples were subjected to electrospray ionisation-mass spectrometry (ESI-MS) analysis employing quadrupole-ion trap as well as high resolution quadrupole time-of-flight (Q-TOF) detectors, and the resulting spectra screened for the detection of polymers in which metal cations have become incorporated into the chain backbone following deprotonation reactions during the ESI process. Adducts formed from such polymer-metal salts were detected in spectra produced from each of the samples under investigation, indicating that the formation, ionisation and detection of polymer-metal salts can be readily achieved under typical ESI-MS experimental conditions. Specifically (under conditions of MeOH containing 0.1 mM sodium acetate) polyAA is most susceptible to the incorporation of metal ions (up to 3 replacements), followed by polyNIPAAm with two replacements and polyDISP with one replacement. The results obtained from the present investigation clearly demonstrate that for synthetic polymers capable of undergoing deprotonation under ESI conditions, the potential detection of polymer-metal salt adducts must be considered during spectral data interpretation to avoid erroneous species assignments.

Optimised matrix-assisted laser desorption/ionisation (MALDI) and electrospray ionisation (ESI) mass spectrometry (MS) methodologies were systematically compared in terms of their relative abilities to identify distinct chemical species present in samples associated with a polymer mechanistic study. In order to perform the investigation, formation processes involved in atom transfer radical polymerisation (ATRP) mediated methyl acrylate (MA) star polymerisations were studied. In addition to the 4-armed ATRP initiator employed in the polymerisations, initiator side-products were found to generate oligomeric chains. At a relatively high monomer to polymer conversion, terminal Br loss was observed in these oligomers; this Br loss was hypothesised to occur via degradative transfer reactions involving the radicals (CH3)2 _ COH, _ CH3 and _ CH2COCH3, which were derived from the acetone used as a solvent in the polymerisations, as well as hydrogen radicals donated by the ligand N,N 0

The generation of star polymers via living polymerization protocols is well documented; however, the impact of midchain radicals (MCRs) on the precise formation pathways under operation in living acrylate star polymerizations is still poorly understood. In the present study, electrospray ionization−mass spectrometry (ESI-MS) technology has been applied to map the products generated in R-group approach reversible addition fragmentation chain transfer (RAFT) methyl acrylate (MA) star polymerizations in order to gain insight into the precise formation pathways under operation in such systems. The polymerizations were conducted at 65 °C using the tetrafunctional RAFT agent 1,2,4,5-tetrakis(2-phenylthioacetylsulfanylmethyl)benzene and 2,2′-azobis(isobutyronitrile) (AIBN) as the thermally decomposing initiator. Initiator fragment derived linear chains, ideal stars, star−star couples, and other terminated star products formed as a result of combination and disproportionation reactions were successfully imaged. A...

The use of silver tetrafluoroborate as a doping salt to achieve efficient and soft desorption/ionization of labile end-group-carrying polystyrene during electrospray ionization is demonstrated. Polystyrene carrying a dithioester end group prepared via reversible addition fragmentation chain transfer (RAFT) chemistry (using the RAFT agent cumyl phenyldithioacetate) as well as a commercial polymer standard prepared by anionic polymerization serve as model compounds. By employing silver tetrafluoroborate as ionization agent, an increase in ion count of more than one order of magnitude was achieved compared to ionization with sodium iodide. Little loss of the end group occurred via elimination of the dithioacid to yield vinyl-terminated polymer. A possible mechanism is given for catalysis of the cleavage reaction in the presence of silver salts. Side-product formation due to thermal or collision induced loss of the dithioester was kept at a minimum under optimized source conditions. Thus, we introduce a novel...

A mass spectrometry analysis has been performed on complex architecture polymeric material produced during reversible addition fragmentation chain transfer (RAFT) polymerizations yielding star polymers. Para-acetoxystyrene (AcOSty) has been polymerized at 60 °C, using azobisisobutyronitrile (AIBN) as the thermally decomposing initiator, in the presence of the R-group approach tetrafunctional RAFT agent (1,2,4,5-tetrakis-(2-phenyl-thioacetyl-sulfanylmethyl)-benzene). In addition to ideal star material, a variety of products unique to this mode of polymerization have been identified. These include star–star couples, stars terminated with initiator fragments, star–star couples terminated with initiator fragments and linear polymers, supporting the notion that these species are responsible for the structured molecular-weight distributions measured for these systems when analyzed via gel permeation chromatography. The analysis begins with a study of AcOSty polymerizing (i) in the absence of any mediating agent and (ii) in the presence of a monofunctional RAFT agent, revealing the mode of termination of propagating poly(AcOSty) radicals as combination and that some ionization biases exist among variants of poly (AcOSty). The interpretation of the mass spectrometry data has been aided by a novel kinetic model of star polymerizations, allowing the rationalization of experimental observations with theoretical expectations. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 1873–1892, 2008

Using proteins as the hydrophilic moiety can dramatically improve the biodegradability and biocompatibility of self-assembled amphiphilic nanoparticles in the field of nanomedicine. In this study, we fabricated and evaluated curcumin loaded albumin-polycaprolactone nanoparticles as a novel drug delivery system for prostate carcinoma therapeutics and compared their performance to poly(methyl methacrylate) (PMMA), a non-degradable and amorphous polymer. The maleimide functionalized poly(epsilon-caprolactone) (PCL) was obtain using ring opening polymerization (ROP) of epsilon-caprolactone where N-(2-hydroxyethyl) maleimide was used as an initiator. The resorbable albumin-polymer conjugate was prepared by conjugating the hydrophobic maleimide-terminated PCL to the hydrophilic bovine serum albumin (BSA) via a simple Michael addition reaction. PMMA was conjugated in a similar manner. The amphiphilic BSA-polymer conjugates can self-assemble into nanoparticles, displaying well-defined structure, prolonged storage stability, and excellent biocompatibility. The BSA nanoparticles, with encapsulated curcumin, exhibited highly enhanced antitumor activity compared to free curcumin. Furthermore, the high efficacy of the curcumin loaded nanoparticles was verified by effectively inhibiting the growth of three-dimensional LNCaP multicellular tumour spheroids. The cytotoxicity was attributed to the efficient cellular uptake of the nanoparticles through caveolic endocytosis. The direct comparison between PCL and the PMMA revealed that drug loading and release as well as cytotoxicity is not significantly affected by the nature of the polymer. However, it seems that nanoparticles based on PMMA penetrate quicker into LNCaP multicellular tumour spheroids thanks to the increased stability. The faster penetration was found to reduce the toxicity of the nanoparticles as evidenced by the lower number of dead cells. In contrast, the fully degradable PCL-based nanoparticles were more efficient in delivering the drug, thus limiting the growth of LNCaP multicellular tumour spheroids. Türkiye Bilimsel Ve Teknolojik Araştırma Kurumu ( Tübitak )

"Smart" polymers and polymer-protein conjugates find a vast array of biomedical applications. Ambient temperature reversible addition fragmentation chain transfer (RAFT) polymerizations conducted in an aqueous environment are a favorable method of choice for the synthesis of these materials; however, information regarding the initiation mechanisms behind these polymerizations-and thus the critical polymer end groups-is lacking. In the current study, high-resolution soft ionization mass spectrometry techniques were used to map the product species generated during ambient temperature gamma-radiation induced RAFT polymerizations of N-isopropylacrylamide (NIPAAm) and acrylic acid (AA) in aqueous media, allowing the generated end groups to be unambiguously established. It was found that trithiocarbonate and *R radicals produced from the radiolysis of the RAFT agent, *OH and *OOH radicals produced from the radiolysis of water, and *H radicals produced from the radiolysis of water, RAFT agent, or monomer were capable of initiating polymerizations and thus contribute toward the generated chain ends. Additionally, thiol terminated chains were formed via degradation of trithiocarbonate end groups. The current study is the first to provide comprehensive mapping of the formation pathways and end group patterns of stimuli-responsive polymers, thus allowing the design and implementation of these materials to proceed in a more tailored fashion.

Reversible addition fragmentation chain transfer (RAFT) mediated methyl acrylate and butyl acrylate polymerizations initiated via 60Co γ-irradiation were investigated using electrospray ionization mass spectrometry (ESI−MS). The polymer end-groupings were mapped using both a hybrid quadrupole-time-of-flight instrument and a quadrupole ion trap mass spectrometer. Three potential initiation processes were identified during the cumyl phenyl dithioacetate (CPDA) mediated acrylate polymerizations. CPDA dissociates into a cumyl radical and a phenyl dithioester radical (Z-group). A set of polymer product ions indicates that a small, yet significant, fraction of the γ-generated phenyl dithioacetate radical contributes to the initiation process. Furthermore, the phenyl dithioacetate group undergoes cleavage to yield thiol end groups that react to form disulfide linkages. Additionally, polymer product ions indicative of a small amount of water radiolysis were observed, and evidence that the butyl acrylate monomer i...

Cholesterol is essential to human health, and its levels are tightly regulated by a balance of synthesis, uptake, and efflux. Cholesterol synthesis requires the actions of more than twenty enzymes to reach the final product, through two alternate pathways. Here we describe a physical and functional interaction between the two terminal enzymes. 24-Dehydrocholesterol reductase (DHCR24) and 7-dehydrocholesterol reductase (DHCR7) coimmunoprecipitate, and when the DHCR24 gene is knocked down by siRNA, DHCR7 activity is also ablated. Conversely, overexpression of DHCR24 enhances DHCR7 activity, but only when a functional form of DHCR24 is used. DHCR7 is important for both cholesterol and vitamin D synthesis, and we have identified a novel layer of regulation, whereby its activity is controlled by DHCR24. This suggests the existence of a cholesterol #x201C;metabolon#x201D;, where enzymes from the same metabolic pathway interact with each other to provide a substrate channeling benefit. We predict that other enzymes in cholesterol synthesis may similarly interact, and this should be explored in future studies.

The delivery of macromolecular platinum drugs into cancerous cells is enhanced by conjugating the polymer to albumin. The monomers N-(2-hydroxypropyl)methacrylamide (HPMA) and Boc protected 1,3-diaminopropan-2-yl acrylate (Ac-DAP-Boc) are copolymerized in the presence of a furan protected maleimide functionalized reversible addition-fragmentation chain transfer (RAFT) agent. The resulting polymer with a composition of P(HPMA14 -co-(Ac-DAP-Boc)9 ) and a molecular weight of Mn = 7600 g mol(-1) (Đ = 1.24) is used as a macromolecular ligand for the conjugation to the platinum drug. Thermogravimetric analysis reveals full conjugation. After deprotection of the maleimide functionality of the polymer, the reactive polymer is conjugated to albumin using the Cys34 functionality. The conjugation is monitored using size exclusion chromatography, MALDI-TOF (matrix assisted laser desorption ionization time-of-flight), and SDS Page (sodium dodecyl sulphate polyacrylamide gel electrophoresis). The polymer-albumin conjugates self-assemble in water into nanoparticles of sizes of around 80 nm thanks to the hydrophobic nature of the platinum drugs. The albumin coated nanoparticles are readily taken up by ovarian cancer cell lines and they show superior toxicity compared to a control sample without protein coating.

A new micelle delivery platform based on albumin coated nanoparticles is able to selectively deliver the payload to cancerous cells while healthy cells remain less affected. The technology is simple and can be used in a one-pot procedure.

Mass spectrometry (MS)-based studies of synthetic polymers often characterise detected polymer components using mass data alone. However when mass-based characterisations are ambiguous, tandem MS (MS/MS) offers a means by which additional analytical information may be collected. This review provides a synopsis of two particularly promising methods of dissociating polymer ions during MS/MS: electron-capture and electron-transfer dissociation (ECD and ETD, respectively). The article opens with a summary of the basic characteristics and operating principles of ECD and ETD, and relates these techniques to other methods of dissociating gas-phase ions, such as collision-induced dissociation (CID). Insights into ECD- and ETD-based MS/MS, gained from studies into proteins and peptides, are then discussed in relation to polymer chemistry. Finally, ECD- and ETD-based studies into various classes of polymer are summarised; for each polymer class, ECD- and ETD-derived data are compared to CID-derived data. These discussions identify ECD and ETD as powerful means by which unique and diagnostically useful polymer ion fragmentation data may be generated, and techniques worthy of increased utilisation by the polymer chemistry community.

Broad-scale mass spectrometric analyses of glycopeptides are constrained by the considerable complexity inherent to glycoproteomics, and techniques are still being actively developed to address the associated analytical difficulties. Here we apply Orbitrap mass analysis and higher-energy C-trap dissociation (HCD) to facilitate detailed insights into the compositions and heterogeneity of complex mixtures of low abundance glycopeptides. By generating diagnostic oxonium product ions at mass measurement errors of