Determination of cell type‐specific proteome signatures of primary human leukocytes,endothelial cells,keratinocytes, hepatocytes,fibroblasts and melanocytes by comparative proteome profiling

Cells gain their functional specialization by different protein synthesis. A lot of knowledge with respect to cell type‐specific proteins has been collected during the last thirty years. This knowledge was built mainly by using antibodies. Nowadays, modern MS, which supports comprehensive proteome analyses of biological samples, may render possible the search for cell type‐specific proteins as well. However, a therefore necessary systematic MS study comprising many different cell types has not been performed until now. Here we present a proteome analysis strategy supporting the automated and meaningful comparison of any biological samples. We have presently applied this strategy to six different primary human cell types, namely leukocytes, endothelial cells, keratinocytes, hepatocytes, fibroblasts, and melanocytes. Comparative analysis of the resulting proteome profiles allowed us to select proteins specifically identified in one of the six cell types and not in any of the five others. Based on these results, we designated cell type‐specific proteome signatures consisting each of six such characteristic proteins. These signatures independently reproduced well‐known marker proteins already established for FACS analyses in addition to novel candidate marker proteins. We applied these signatures for the interpretation of proteome profiles obtained from the analyses of hepatocellular carcinoma‐associated tissue homogenates and normal liver tissue homogenates. The identification of members of the above described signatures gave us an indication of the presence of characteristic cells in the diseased tissues and thus supported the interpretation of the proteomics data of these complex biological samples.     

EFFECT OF UVA AND BLUE LIGHT ON PORPHYRIN BIOSYNTHESIS IN EPIDERMAL CELLS*

Abstract— To study porphyrin biosynthesis in normal human keratinocytes and A431 cells derived from human epidermoid carcinoma, cultured cells were incubated with delta-aminolevulinic acid (ALA), the precursor of porphyrin synthesis, and accumulation of porphyrins was measured spectrofluorometrically. Both human keratinocytes and A431 cells accumulated porphyrins in a time-dependent and a dose-dependent fashion. Protoporphyrin was the predominant porphyrin accumulated by both cell types. Porphyrin accumulation was enhanced by Ca Mg ethylene-diaminetetraacetic acid, a ferrochelatase inhibitor, and the enhancement was reversed by the addition of iron, suggesting the utilization of iron by ferrochelatase. The effect of light on porphyrin accumulation was evaluated by exposing the ALA-loaded A431 cells to ultraviolet-A (UVA) and blue light radiation, followed by continued incubation with ALA for 2–48 h. There was an enhancement of porphyrin accumulation 2–48 h after the radiation as compared with nonirradiated controls. Consistent with this finding, ferrochelatase activity decreased in these cells at 24 h and 48 h. These data demonstrate that human keratinocytes and A431 cells are capable of porphyrin biosynthesis, and that exposure of porphyrin-containing A431 cells to light, which includes the Soret band spectrum, decreases the ferrochelatase activity, which is responsible, at least in part, for the further increase in porphyrin level.     

Proteomics Analysis of T Lymphocytes Damage Induced by Ionizing Irradiation

Human T lymphocytes were found to be highly radiosensitive and complex cellular responses including apoptosis could be induced upon exposure to X‐ray irradiation. However, the mechanism of apoptosis associated with irradiation was not clear. In this study, a proteomic method was applied to investigation on alteration of proteome of human T‐lymphocyte cells after irradiation. The Jurkat cells were irradiated with 4 Gy X‐ray and the cell lysates were collected at different times after irradiation (6, 12, 18, 24 and 48 h). The whole proteins were separated and quantified by two‐dimensional fluorescence difference gel electrophoresis, and then the differentially expressed proteins were identified by mass spectrometry. 4 proteins exhibited significant irradiation‐induced difference in abundance, including L‐plastin, bifunctional purine biosynthesis protein, tubulin beta chain, beta‐actin. Differentially expressed proteins were reported to be directly or indirectly involved in the function of human T lymphocyte. Thus, this study might provide clues to identify proteins with biological significance related to irradiation.     

A Non‐Invasive NMR Method Based on Histidine Imidazoles to Analyze the pH‐Modulation of Protein–Nucleic Acid Interfaces

A useful ²J(N?H) coupling‐based NMR spectroscopic approach is proposed to unveil, at the molecular level, the contribution of the imidazole groups of histidines from RNA/DNA‐binding proteins on the modulation of binding to nucleic acids by pH. Such protonation/deprotonation events have been monitored on the single His96 located at the second RNA/DNA recognition motif (RRM2) of T‐cell intracellular antigen‐1 (TIA‐1) protein. The pK_a values of the His96 ionizable groups were substantially higher in the complexes with short U‐rich RNA and T‐rich DNA oligonucleotides than those of the isolated TIA‐1 RRM2. Herein, the methodology applied to determine changes in pK_a of histidine side chains upon DNA/RNA binding, gives valuable information to understand the pH effect on multidomain DNA/RNA‐binding proteins that shuttle among different cellular compartments.     

Deficient Nucleotide Excision Repair in Squamous Cell Carcinoma Cells

Squamous cell carcinomas (SCCs) are associated with ultraviolet radiation and multiple genetic changes, but the mechanisms leading to genetic instability are unclear. SCC cell lines were compared to normal keratinocytes for sensitivity to ultraviolet radiation, DNA repair kinetics and DNA repair protein expression. Relative to normal keratinocytes, four SCC cell lines were all variably sensitive to ultraviolet radiation and, except for the SCC25 cell line, were deficient in global repair of cyclobutane pyrimidine dimers, although not 6‐4 photoproducts. Impaired DNA repair of cyclobutane pyrimidine dimers was associated with reduced mRNA expression from XPC but not DDB2 genes which each encode key DNA damage recognition proteins. However, levels of XPC or DDB2 proteins or both were variably reduced in repair‐deficient SCC cell lines. p53 levels did not correlate with DNA repair activity or with XPC and DDB2 levels, but p63 levels were deficient in cell lines with reduced global repair. Repair‐proficient SCC25 cells depleted of p63 lost XPC expression, early global DNA repair activity and UV resistance. These results demonstrate that some SCC cell lines are deficient in global nucleotide excision repair and support a role for p63 as a regulator of nucleotide excision repair in SCCs.     

Antioxidant,DNA interaction,molecular docking and cytotoxicity studies of aminoethylpiperazine‐containing macrocyclic binuclear copper(II) complexes

A series of new macrocyclic binuclear copper(II) complexes of the type [Cu₂L^1–5(ClO₄)](ClO₄) ( 1 – 5 ) were synthesized by template condensation between precursor compounds 2,6‐bis(4‐aminoethylpiperazin‐1‐ylmethyl)‐4‐substituted phenols and 2,6‐diformyl‐4‐substituted phenols. The synthesized precursors and complexes were characterized using regular physicochemical techniques. The rate constant values obtained for the hydrolysis of 4‐nitrophenylphosphate were in the range 1.83 × 10⁻²–4.19 × 10⁻² min⁻¹. Antioxidant studies against 2,2′‐diphenyl‐1‐picrylhydrazyl revealed the antioxidant potency of the synthesized complexes. Binding studies of the complexes with calf thymus DNA were conducted using electronic, viscometric and voltammetric techniques, and the obtained results suggested a non‐covalent groove mode of binding. The oxidative cleavage of pBR322 DNA in the presence of co‐reactant H₂O₂ and radical scavengers showed single strand scission and involvement of H₂O₂ radical in the cleavage process. Molecular docking studies were performed to insert complexes into the crystal structures of 1BNA and VEGFR kinase at active sites to determine the possible binding mode and predominant binding interactions. In vitro cytotoxicity of the complexes was tested against human epidermoid carcinoma cells (A431) by MTT assay, which revealed the effective anticancer activity of the complexes. Live cell and fluorescent imaging of A431 cells showed that the complexes induce cell death through apoptosis.     

Trident Nano-Indexing the Proteomics Table: Next-Version Clustering of Iron Carbide NPs and Protein Corona

Protein corona composition and precise physiological understanding of differentially expressed proteins are key for identifying disease biomarkers. In this report, we presented a distinctive quantitative proteomics table of molecular cell signaling differentially expressed proteins of corona that formed on iron carbide nanoparticles (NPs). High-performance liquid chromatography/electrospray ionization coupled with ion trap mass analyzer (HPLC/ESI-Orbitrap) and MASCOT helped quantify 142 differentially expressed proteins. Among these proteins, 104 proteins showed upregulated behavior and 38 proteins were downregulated with respect to the control, whereas 48, 32 and 24 proteins were upregulated and 8, 9 and 21 were downregulated CW (control with unmodified NPs), CY (control with modified NPs) and WY (modified and unmodified NPs), respectively. These proteins were further categorized on behalf of their regularity, locality, molecular functionality and molecular masses using gene ontology (GO). A STRING analysis was used to target the specific range of proteins involved in metabolic pathways and molecular processing in different kinds of binding functionalities, such as RNA, DNA, ATP, ADP, GTP, GDP and calcium ion bindings. Thus, this study will help develop efficient protocols for the identification of latent biomarkers in early disease detection using protein fingerprints.     

RNA–DNA Chimeras in the Context of an RNA World Transition to an RNA/DNA World

The RNA world hypothesis posits that DNA and proteins were later inventions of early life, or the chemistry that gave rise to life. Most scenarios put forth for the emergence of DNA assume a clean separation of RNA and DNA polymer, and a smooth transition between RNA and DNA. However, based on the reality of “clutter” and lack of sophisticated separation/discrimination mechanisms in a protobiological (and/or prebiological) world, heterogeneous RNA–DNA backbone containing chimeric sequences could have been common—and have not been fully considered in models transitioning from an RNA world to an RNA–DNA world. Herein we show that there is a significant decrease in Watson–Crick duplex stability of the heterogeneous backbone chimeric duplexes that would impede base‐pair mediated interactions (and functions). These results point to the difficulties for the transition from one homogeneous system (RNA) to another (RNA/DNA) in an RNA world with a heterogeneous mixture of ribo‐ and deoxyribonucleotides and sequences, while suggesting an alternative scenario of prebiological accumulation and co‐evolution of homogeneous systems (RNA and DNA).     

Myofibroblasts are important contributors to human hepatocellular carcinoma: Evidence for tumor promotion by proteome profiling

Liver cancer typically occurs with a background of chronic fibrosis, characterized by the accumulation of myofibroblast‐like cells. We performed 2D‐PAGE‐based comparative analyses with the aim to identify proteins expressed in human hepatocellular carcinoma (HCC) tissue but not in neighboring healthy liver tissue, and to make out which cell types are responsible for the expression of proteins most characteristic for HCC. LC‐MS/MS analysis of the most striking spots identified proteins that were mainly related to myofibroblast‐like cells. To gain more insights into the role of these cells in their contribution to HCC, we isolated myofibroblast‐like cells as well as hepatocytes, both derived from HCC tissues, and subjected them to proteome profiling based on shotgun experiments. Comparative analysis, also referring to proteome profiles of other cell types previously investigated by us, pointed again to a marked contribution of myofibroblast‐like cells to HCC. Intriguingly, secretome analysis of these cells identified several growth factors that may act as tumor promoters and several proteins that have been described as potential biomarkers for HCC including dickkopf‐1, connective tissue growth factor, and CXCL1. Other biomarker candidates presently identified in the secretome of myofibroblasts, including lipocalin‐1 and pappalysin‐1, may be selected for future clinical validation. The identification of myofibroblast‐like cells as important source of tumor‐promoters may open new avenues to therapeutic intervention by targeting these stroma cells in addition to the cancer cells.     

Segmental,Domain‐Selective Perdeuteration and Small‐Angle Neutron Scattering for Structural Analysis of Multi‐Domain Proteins

Multi‐domain proteins play critical roles in fine‐tuning essential processes in cellular signaling and gene regulation. Typically, multiple globular domains that are connected by flexible linkers undergo dynamic rearrangements upon binding to protein, DNA or RNA ligands. RNA binding proteins (RBPs) represent an important class of multi‐domain proteins, which regulate gene expression by recognizing linear or structured RNA sequence motifs. Here, we employ segmental perdeuteration of the three RNA recognition motif (RRM) domains in the RBP TIA‐1 using Sortase A mediated protein ligation. We show that domain‐selective perdeuteration combined with contrast‐matched small‐angle neutron scattering (SANS), SAXS and computational modeling provides valuable information to precisely define relative domain arrangements. The approach is generally applicable to study conformational arrangements of individual domains in multi‐domain proteins and changes induced by ligand binding.     

Segmental,Domain‐Selective Perdeuteration and Small‐Angle Neutron Scattering for Structural Analysis of Multi‐Domain Proteins

Multi‐domain proteins play critical roles in fine‐tuning essential processes in cellular signaling and gene regulation. Typically, multiple globular domains that are connected by flexible linkers undergo dynamic rearrangements upon binding to protein, DNA or RNA ligands. RNA binding proteins (RBPs) represent an important class of multi‐domain proteins, which regulate gene expression by recognizing linear or structured RNA sequence motifs. Here, we employ segmental perdeuteration of the three RNA recognition motif (RRM) domains in the RBP TIA‐1 using Sortase A mediated protein ligation. We show that domain‐selective perdeuteration combined with contrast‐matched small‐angle neutron scattering (SANS), SAXS and computational modeling provides valuable information to precisely define relative domain arrangements. The approach is generally applicable to study conformational arrangements of individual domains in multi‐domain proteins and changes induced by ligand binding.     

Establishment of A431 cell membrane chromatography-RPLC method for screening target components from Radix Caulophylli

We describe here an analytical method of A431 cell membrane chromatography (A431/CMC) (CMC, cell membrane chromatography) combined with RPLC for recognition, separation, and identification of target components from traditional Chinese medicines (TCMs) Radix Caulophylli. The A431 cells with high expressed epidermal growth factor receptor (EGFR) were used to prepare the stationary phase in the CMC model. Retention fractions on the A431-CMC model were collected using an automated fraction collection and injection module (FC/I). Each fraction was analyzed by RPLC under the optimized conditions. Gefitinib and erlotinib were used as standard compounds to investigate the suitability and reliability of the A431 cell membrane chromatography-RPLC method prior to screening target component from Radix Caulophylli total alkaloids. The results indicated that caulophine and taspine were the target component acting on the epidermal growth factor receptor. This method could be an efficient way in drug discovery using natural medicinal herbs as a source of novel compounds.