Analysis of the peptides detected in atopic dermatitis and various inflammatory diseases patients-derived sera.

Serum proteomics has been applied for the discovery and analysis of biomarkers related to human disease. Serum is an optimal source to identify proteins derived from diseased-tissue compartments. We recently established an integrative method to analyze highly basic proteins that remain unresolved by the general 2D-PAGE method. In this follow-up study, we successfully detected several disease-associated proteins from sera samples obtained from patients with atopic dermatitis (AD). After proteomic analyses, target proteins were validated from AD patient-derived sera using ELISA or Western blotting methods We detected zinc finger CCHC domain containing 10 (ZCCHC10), peptidoglycan recognition protein L (PGRP-L), kininogen, α-1-antitrypsin, and hornerin proteins that are dysregulated in AD patient sera samples, which suggest effective approaches to methodologically analyze the serum proteome. Thus, the integrated proteomic method approach described here could be applicable for the detection of proteins associated with other human diseases. Our present study provides new insights into optimized serum proteomic techniques to understand systemic events of AD.

Evaluation of the structural, physicochemical, and biological characteristics of SB4, a biosimilar of etanercept.

A biosimilar is a biological medicinal product that is comparable to a reference medicinal product in terms of quality, safety, and efficacy. SB4 was developed as a biosimilar to Enbrel® (etanercept) and was approved as Benepali®, the first biosimilar of etanercept licensed in the European Union (EU). The quality assessment of SB4 was performed in accordance with the ICH comparability guideline and the biosimilar guidelines of the European Medicines Agency and Food and Drug Administration. Extensive structural, physicochemical, and biological testing was performed with state-of-the-art technologies during a side-by-side comparison of the products. Similarity of critical quality attributes (CQAs) was evaluated on the basis of tolerance intervals established from quality data obtained from more than 60 lots of EU-sourced and US-sourced etanercept. Additional quality assessment was focused on a detailed investigation of immunogenicity-related quality attributes, including hydrophobic variants, high-molecular-weight (HMW) species, N-glycolylneuraminic acid (NGNA), and α-1,3-galactose. This comprehensive characterization study demonstrated that SB4 is highly similar to the reference product, Enbrel®, in structural, physicochemical, and biological quality attributes. In addition, the levels of potential immunogenicity-related quality attributes of SB4 such as hydrophobic variants, HMW aggregates, and α-1,3-galactose were less than those of the reference product.

An antimicrobial protein, lactoferrin exists in the sweat: proteomic analysis of sweat.

The main function of the eccrine gland has been considered to be thermoregulation. Recently, it has been reported that antimicrobial peptides including cathelicidin and dermcidin exist in the sweat. Lactoferrin is found in body fluids such as milk tears and saliva. It is known as a component of host defense against infection and inflammation. In this study, we explored whether lactoferrin is produced by eccrine glands, thereby establishing its potential role in the skin defense. By immunohistochemistry, lactoferrin was detected in eccrine glands of normal human skin. In Western blot analysis, lactoferrin was found in sweat and skin surface substances obtained from healthy volunteers. By proteomic analysis, lactoferrin and other antimicrobial peptides were detected in sweat. In addition, we measured the concentration of lactoferrin in sweat by enzyme-linked immunosorbent assay. These findings suggest that lactoferrin may contribute to skin defense against infection through its secretion in sweat.

An analysis of an interactome for apoptosis factor, Ei24/PIG8, using the inducible expression system and shotgun proteomics.

ei24 (etoposide-induced 2.4 kb transcript, also designated PIG8 (p53-induced gene 8)), is a DNA damage response gene involved in growth suppression and apoptosis. ei24 gene expression is specifically induced by wild type p53, and its overexpression suppresses cell growth by inducing apoptotic cell death. Generally, the protein-protein interaction is known to regulate their targets, as well as to modify cell fates. In this study, using the established NIH/3T3, oncogenic H-Ras/G12V transformed NIH/3T3, and B16F10 cells, which are expressing mouse Ei24 proteins under the tight control of expression by doxycycline, a proteomic screening was conducted to identify the binding partners for Ei24. Immunoprecipitation of mEi24 and associated proteins was performed using the mEi24 expressing cell lysates. Isolated immuno-complexes were resolved by SDS-PAGE and analyzed by liquid chromatography tandem mass spectrometry. There were 61 novel potential mEi24 interacting proteins identified, among which are NIH/3T3/mEi24, H-Ras/G12V/NIH/3T3/mEi24, and B16F10/mEi24 cells; however, some mEi24 interacting proteins were specific to two NIH/3T3 related cells and to B16F10/mEi24 cells. This approach led to the identification of many interacting partners, and the discovery of these associated proteins will lead to a better understanding of the mechanisms underlying the physiological and cell biological roles of Ei24.

Regulation of mouse steroidogenesis by WHISTLE and JMJD1C through histone methylation balance.

The dynamic exchange of histone lysine methylation status by histone methyltransferases and demethylases has been previously implicated as an important factor in chromatin structure and transcriptional regulation. Using immunoaffinity TAP analysis, we purified the WHISTLE-interacting protein complexes, which include the heat shock protein HSP90α and the jumonji C-domain harboring the histone demethylase JMJD1C. In this study, we demonstrate that JMJD1C specifically demethylates histone H3K9 mono- and di-methylation, and mediates transcriptional activation. We also provide evidence suggesting that both WHISTLE and JMJD1C performs functions in the development of mouse testes by regulating the expression of the steroidogenesis marker, p450c17, via SF-1-mediated transcription. Furthermore, we demonstrate that WHISTLE is recruited to the p450c17 promoter via SF-1 and represses the transcription of prepubertal stages of steroidogenesis, after which JMJD1C replaces WHISTLE and activates the expression of target genes via SF-1-mediated interactions. Our results demonstrate that the histone methylation balance mediated by HMTase WHISTLE and demethylase JMJD1C perform a transcriptional regulatory function in mouse testis development.

Towards profiling the gene expression of tyrosinase-induced melanogenesis in HEK293 cells: a functional DNA chip microarray and interactomics studies.

The overexpression of a single tyrosinase gene can induce conspicuous pigmentation in nonpigmented cells. We hypothesized that some unknown tyrosinase-associated genes are simultaneously regulated by melanin synthesis. To improve understanding of melanogenesis and tyrosinase-associated functions, we attempted to profile the genes that are altered during melanin production in HEK293 cells by using a functional DNA chip microarray. The candidate genes were obtained based on significance analysis of microarray (SAM) and further computational prediction via protein-protein interaction (PPI) mapping suggested that newly detected hub genes were involved in melanogenesis. PPI mapping using bioinformatic tools revealed 8 genes that formed an interaction hub. The yeast two-hybridization results suggested some candidate genes could interact with tyrosinase. The present study provides information to further understand the complex factors associated with tyrosinase-induced melanogenesis and apoptosis. The approach of combining expression data analysis and predicted protein interaction partners can help identify genes involved in pigmentation.

A cross-talk between oncogenic Ras and tumor suppressor PTEN through FAK Tyr861 phosphorylation in NIH/3T3 mouse embryonic fibroblasts.

Although Ras is a potent oncogene, its tumorigenicity depends on cellular context and cooperative events. Tumor suppressor PTEN is the most important negative regulator of the cell-survival signaling pathway initiated by phosphoinositide 3-OH kinase. Previously, we established various NIH/3T3 cells expressing H-Ras mutant proteins. This report shows that expression of PTEN is suppressed by the oncogenic H-Ras at its protein and transcript levels as well as by oncogenic K- and N-Ras. This activity of oncogenic Ras is mediated by Raf-1/Erk/MEK signaling pathway. In our previous reports, FAK Y(861) phosphorylation is higher in H-Ras transformed NIH/3T3 cells. In this report, level of FAK pY(861) was examined in Ras mutant cell lines. By generating wild-type PTEN, lipid phosphatase-deficient PTEN and activity-inert PTEN-inducible cell lines in the background of oncogenic H-Ras stable expression in NIH/3T3 cells, we show level of FAK pY(861) is decreased by protein phosphatase activity of PTEN.