Protein profiling of human lung telocytes and microvascular endothelial cells using iTRAQ quantitative proteomics

Telocytes (TCs) are described as a particular type of cells of the interstitial space ( www.telocytes.com ). Their main characteristics are the very long telopodes with alternating podoms and podomers. Recently, we performed a comparative proteomic analysis of human lung TCs with fibroblasts, demonstrating that TCs are clearly a distinct cell type. Therefore, the present study aims to reinforce this idea by comparing lung TCs with endothelial cells (ECs), since TCs and ECs share immunopositivity for CD34. We applied isobaric tag for relative and absolute quantification (iTRAQ) combined with automated 2‐D nano‐ESI LC‐MS/MS to analyse proteins extracted from TCs and ECs in primary cell cultures. In total, 1609 proteins were identified in cell cultures. 98 proteins (the 5th day), and 82 proteins (10th day) were confidently quantified (screened by two‐sample t‐test, P < 0.05) as up‐ or down‐regulated (fold change >2). We found that in TCs there are 38 up‐regulated proteins at the 5th day and 26 up‐regulated proteins at the 10th day. Bioinformatics analysis using Panther revealed that the 38 proteins associated with TCs represented cellular functions such as intercellular communication (via vesicle mediated transport) and structure morphogenesis, being mainly cytoskeletal proteins and oxidoreductases. In addition, we found 60 up‐regulated proteins in ECs e.g.: cell surface glycoprotein MUC18 (15.54‐fold) and von Willebrand factor (5.74‐fold). The 26 up‐regulated proteins in TCs at 10th day, were also analysed and confirmed the same major cellular functions, while the 56 down‐regulated proteins confirmed again their specificity for ECs. In conclusion, we report here the first extensive comparison of proteins from TCs and ECs using a quantitative proteomics approach. Our data show that TCs are completely different from ECs. Protein expression profile showed that TCs play specific roles in intercellular communication and intercellular signalling. Moreover, they might inhibit the oxidative stress and cellular ageing and may have pro‐proliferative effects through the inhibition of apoptosis. The group of proteins identified in this study needs to be explored further for the role in pathogenesis of lung disease.     

Cariprazine (RGH-188), a potent D3/D2 dopamine receptor partial agonist, binds to dopamine D3 receptors in vivo and shows antipsychotic-like and procognitive effects in rodents

We investigated the in vivo effects of orally administered cariprazine (RGH-188; trans-N-{4-[2-[4-(2,3-dichlorophenyl)-piperazin-1-yl]-ethyl]-cyclohexyl}-N′,N′-dimethyl-urea), a D₃/D₂ dopamine receptor partial agonist with ∼10-fold preference for the D₃ receptor. Oral bioavailability of cariprazine at a dose of 1 mg/kg in rats was 52% with peak plasma concentrations of 91 ng/mL. Cariprazine 10 mg/kg had good blood-brain barrier penetration, with a brain/plasma AUC ratio of 7.6:1. In rats, cariprazine showed dose-dependent in vivo displacement of [³H](+)-PHNO, a dopamine D₃ receptor-preferring radiotracer, in the D₃ receptor-rich region of cerebellar lobules 9 and 10. Its potent inhibition of apomorphine-induced climbing in mice (ED₅₀ = 0.27 mg/kg) was sustained for 8 h. Cariprazine blocked amphetamine-induced hyperactivity (ED₅₀ = 0.12 mg/kg) and conditioned avoidance response (CAR) (ED₅₀ = 0.84 mg/kg) in rats, and inhibited the locomotor-stimulating effects of the noncompetitive NMDA antagonists MK-801 (ED₅₀ = 0.049 mg/kg) and phencyclidine (ED₅₀ = 0.09 mg/kg) in mice and rats, respectively. It reduced novelty-induced motor activity of mice (ED₅₀ = 0.11 mg/kg) and rats (ED₅₀ = 0.18 mg/kg) with a maximal effect of 70% in both species. Cariprazine produced no catalepsy in rats at up to 100-fold dose of its CAR inhibitory ED₅₀ value. Cariprazine 0.02-0.08 mg/kg significantly improved the learning performance of scopolamine-treated rats in a water-labyrinth learning paradigm. Though risperidone, olanzapine, and aripiprazole showed antipsychotic-like activity in many of these assays, they were less active against phencyclidine and more cataleptogenic than cariprazine, and had no significant effect in the learning task. The distinct in vivo profile of cariprazine may be due to its higher affinity and in vivo binding to D₃ receptors versus currently marketed typical and atypical antipsychotics.     

A sphingosine-dependent protein kinase that specifically phosphorylates 14-3-3 (SDK1) is identified as the kinase domain of PKCdelta: a preliminary note

A specific protein kinase that phosphorylates Ser60, Ser59, or Ser58 of 14-3-3beta, eta, or zeta, respectively, only in the presence of sphingosine (Sph) or N,N-dimethyl-Sph (DMS), was termed "sphingosine-dependent protein kinase-1" (SDK1) [J. Biol. Chem. 273(34) (1998) 21834]. We have now identified SDK1 as a protein having the same amino acid sequence as in the C-terminal-half kinase domain of PKCdelta, with approximately 40 kDa molecular mass, based on large-scale purification of a protein from rat liver, and partial sequence using three different combinations of LC-MS or LC-MS/MS with respective search engine. PKCdelta did not display any SDK1 activity and PKCdelta activity was inhibited by Sph and DMS. However, strong SDK1 activity, only in the presence of Sph or DMS, became detectable when PKCdelta was incubated with caspase-3, which releases the approximately 40 kDa kinase domain.     

Simultaneous measurement of plasma vitamin D(3) metabolites, including 4β,25-dihydroxyvitamin D(3), using liquid chromatography-tandem mass spectrometry

Simultaneous and accurate measurement of circulating vitamin D metabolites is critical to studies of the metabolic regulation of vitamin D and its impact on health and disease. To that end, we have developed a specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method that permits the quantification of major circulating vitamin D₃ metabolites in human plasma. Plasma samples were subjected to a protein precipitation, liquid–liquid extraction, and Diels–Alder derivatization procedure prior to LC–MS/MS analysis. Importantly, in all human plasma samples tested, we identified a significant dihydroxyvitamin D₃ peak that could potentially interfere with the determination of 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] concentrations. This interfering metabolite has been identified as 4β,25-dihydroxyvitamin D₃ [4β,25(OH)₂D₃] and was found at concentrations comparable to 1α,25(OH)₂D₃. Quantification of 1α,25(OH)₂D₃ in plasma required complete chromatographic separation of 1α,25(OH)₂D₃ from 4β,25(OH)₂D₃. An assay incorporating this feature was used to simultaneously determine the plasma concentrations of 25OHD₃, 24R,25(OH)₂D₃, 1α,25(OH)₂D₃, and 4β,25(OH)₂D₃ in healthy individuals. The LC–MS/MS method developed and described here could result in considerable improvement in quantifying 1α,25(OH)₂D₃ as well as monitoring the newly identified circulating metabolite, 4β,25(OH)₂D₃.     

d-Ribose glycates β2-microglobulin to form aggregates with high cytotoxicity through a ROS-mediated pathway

β₂-Microglobulin (β₂M) modified with advanced glycation end products (AGEs) is a major component of the amyloid deposits in hemodialysis-associated amyloidosis (HAA). However, the effect of glycation on the misfolding and aggregation of β₂M has not been studied so far. Here we examine the molecular mechanism of aggregate formation of HAA-related ribosylated β₂M in vitro. We find that the glycating agent d-ribose interacts with human β₂M to generate AGEs that form aggregates in a time-dependent manner. Ribosylated β₂M molecules are highly oligomerized compared with unglycated β₂M, and have granular morphology. Furthermore, such ribosylated β₂M aggregates show significant cytotoxicity to both human SH-SY5Y neuroblastoma and human foreskin fibroblast FS2 cells and induce intracellular reactive oxygen species (ROS). Presence of the antioxidant N-acetylcysteine (1.0 mM) attenuated intracellular ROS and prevented cell death induction in both SH-SY5Y and FS2 cells, indicating that the cytotoxicity of ribosylated β₂M aggregates depends on a ROS-mediated pathway in both cell lines. In other words, d-ribose reacts with β₂M and induces the ribosylated protein to form granular aggregates with high cytotoxicity through a ROS-mediated pathway. These findings suggest that ribosylated β₂M aggregates could contribute to the dysfunction and death of cells and could play an important role in the pathogenesis of β₂M-associated diseases such as HAA.     

Mass spectrometric characterization of the isoforms in Escherichia coli recombinant DNA-derived interferon alpha-2b

The isoforms Iso-2, Iso-3, and Iso-4 of Escherichia coli-derived recombinant human interferon alpha-2b (rhIFN α-2b), generated by posttranslational modifications of the protein during fermentation, present a major problem in terms of purification and the yield of the drug substance. We report here the structural characterization of these isoforms by mass spectrometry (MS) methods. An extensive MS study was conducted on Iso-4, which is composed of up to 75% of the in-process IFN, and on the native rhIFN α-2b. The trypsin-digested peptide mixtures generated from the two samples were analyzed by liquid chromatography (LC)–MS, and targeted peptides were further studied by LC–tandem MS (triple quadrupole mass spectrometer), high-resolution MSⁿ (LTQ Orbitrap), and matrix-assisted laser desorption/ionization MS (MALDI–MS). The structure of Iso-4 was elucidated as a novel pyruvic acid ketimine derivative of the N-terminal cysteine (Cys1) of IFN α-2b, where the disulfide bond between Cys1 and Cys98 was fully reduced and the other disulfide bond pair, Cys29-ss-Cys138, was partially reduced. Similarly, Iso-2 was identified as a correctly disulfide-folded rhIFN α-2b with acetylation on Cys1, and Iso-3 was identified as an S-glutathionylated form (Cys98) of partially reduced rhIFN α-2b that was pyruvated on Cys1. Based on the characterization work, a reproducible conversion procedure was successfully implemented to convert Iso-4 to rhIFN α-2b.     

In-depth structural characterization of Kadcyla® (ado-trastuzumab emtansine) and its biosimilar candidate

The biopharmaceutical industry has become increasingly focused on developing biosimilars as less expensive therapeutic products. As a consequence, the regulatory approval of 2 antibody-drug conjugates (ADCs), Kadcyla® and Adcetris® has led to the development of biosimilar versions by companies located worldwide. Because of the increased complexity of ADC samples that results from the heterogeneity of conjugation, it is imperative that close attention be paid to the critical quality attributes (CQAs) that stem from the conjugation process during ADC biosimilar development process. A combination of physicochemical, immunological, and biological methods are warranted in order to demonstrate the identity, purity, concentration, and activity (potency or strength) of ADC samples. As described here, we performed extensive characterization of a lysine conjugated ADC, ado-trastuzumab emtansine, and compared its CQAs between the reference product (Kadcyla®) and a candidate biosimilar. Primary amino acid sequences, drug-to-antibody ratios (DARs), conjugation sites and site occupancy data were acquired and compared by LC/MS methods. Furthermore, thermal stability, free drug content, and impurities were analyzed to further determine the comparability of the 2 ADCs. Finally, biological activities were compared between Kadcyla® and biosimilar ADCs using a cytotoxic activity assay and a HER2 binding assay. The in-depth characterization helps to establish product CQAs, and is vital for ADC biosimilars development to ensure their comparability with the reference product, as well as product safety.