Young-Ki Paik

Guidelines for the next 10 years of proteomics

In the last ten years, the field of proteomics has expanded at a rapid rate. A range of exciting new technology has been developed and enthusiastically applied to an enormous variety of biological questions. However, the degree of stringency required in proteomic data generation and analysis appears to have been underestimated. As a result, there are likely to be numerous published findings that are of questionable quality, requiring further confirmation and/or validation. This manuscript outlines a number of key issues in proteomic research, including those associated with experimental design, differential display and biomarker discovery, protein identification and analytical incompleteness. In an effort to set a standard that reflects current thinking on the necessary and desirable characteristics of publishable manuscripts in the field, a minimal set of guidelines for proteomics research is then described. These guidelines will serve as a set of criteria which editors of PROTEOMICS will use for assessment of future submissions to the Journal.

Chemical structure and biological activity of the Caenorhabditis elegans dauer-inducing pheromone

Pheromones are cell type-specific signals used for communication between individuals of the same species. When faced with overcrowding or starvation, Caenorhabditis elegans secrete the pheromone daumone, which facilitates communication between individuals for adaptation to adverse environmental stimuli. Daumone signals C. elegans to enter the dauer stage, an enduring and non-ageing stage of the nematode life cycle with distinctive adaptive features and extended life. Because daumone is a key regulator of chemosensory processes in development and ageing, the chemical identification of daumone is important for elucidating features of the daumone-mediated signalling pathway. Here we report the isolation of natural daumone from C. elegans by large-scale purification, as well as the total chemical synthesis of daumone. We present the stereospecific chemical structure of purified daumone, a fatty acid derivative. We demonstrate that both natural and chemically synthesized daumones equally induce dauer larva formation in C. elegans (N2 strain) and certain dauer mutants, and also result in competition between food and daumone. These results should help to elucidate the daumone-mediated signalling pathway, which might in turn influence ageing and obesity research and the development of antinematodal drugs.

The human proteome project: current state and future direction.

After the successful completion of the Human Genome Project, the Human Proteome Organization has recently officially launched a global Human Proteome Project (HPP), which is designed to map the entire human protein set. Given the lack of protein-level evidence for about 30% of the estimated 20,300 protein-coding genes, a systematic global effort will be necessary to achieve this goal with respect to protein abundance, distribution, subcellular localization, interaction with other biomolecules, and functions at specific time points. As a general experimental strategy, HPP research groups will use the three working pillars for HPP: mass spectrometry, antibody capture, and bioinformatics tools and knowledge bases. The HPP participants will take advantage of the output and cross-analyses from the ongoing Human Proteome Organization initiatives and a chromosome-centric protein mapping strategy, termed C-HPP, with which many national teams are currently engaged. In addition, numerous biologically driven and disease-oriented projects will be stimulated and facilitated by the HPP. Timely planning with proper governance of HPP will deliver a protein parts list, reagents, and tools for protein studies and analyses, and a stronger basis for personalized medicine. The Human Proteome Organization urges each national research funding agency and the scientific community at large to identify their preferred pathways to participate in aspects of this highly promising project in a HPP consortium of funders and investigators.

The Chromosome-Centric Human Proteome Project for cataloging proteins encoded in the genome

The Chromosome-Centric Human Proteome Project for cataloging proteins encoded in the genome

Blocking tumor cell migration and invasion with biphenyl isoxazole derivative KRIBB3, a synthetic molecule that inhibits Hsp27 phosphorylation.

Cell migration is a prerequisite for cancer invasion and metastasis, suggesting cell motility as a potential therapeutic target for cancer treatment. A synthetic library was screened to identify inhibitors of tumor cell migration. From this, we discovered that CAC-1098 (aurintricarboxylic acid) and CBI-0997 (5-(2,4-dimethoxy-5-ethylphenyl)-4-(4-bromophenyl) isoxazole) inhibited migration of MDA-MB-231 cells with IC50 = 5 and 50 nm, respectively. We synthesized KRIBB3 (5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl) isoxazole) by replacing the bromide group of CBI-0997 with a methoxyl group. Like CBI-0997, KRIBB3 has anti-migratory and anti-invasive activities in MDA-MB-231 cells. Because KRIBB3 has a better drug-like structure, we focused our effort on further understanding its anti-migratory mechanism. Biotinyl-KRIBB3 was synthesized as an affinity probe for identification of KRIBB3-binding proteins. Using affinity chromatography, we identified Hsp27 as a target protein of KRIBB3 in vitro. Treatment of MDA-MB-231 cells with phorbol 12-myristate 13-acetate induced protein kinase C-dependent phosphorylation of Hsp27 and tumor cell migration. In contrast, treatment of MDA-MB-231 cells with KRIBB3 blocked phorbol 12-myristate 13-acetate-induced phosphorylation of Hsp27 and tumor cell migration. Furthermore, overexpression of Hsp27 antagonized the inhibitory effect of KRIBB3 on tumor cell invasion, and knockdown of Hsp27 using small interfering RNA inhibited tumor cell migration. Overall, our results demonstrate that KRIBB3 inhibits tumor cell migration and invasion by blocking protein kinase C-dependent phosphorylation of Hsp27 through its direct binding to Hsp27.

Standard guidelines for the chromosome-centric human proteome project.

The objective of the international Chromosome-Centric Human Proteome Project (C-HPP) is to map and annotate all proteins encoded by the genes on each human chromosome. The C-HPP consortium was established to organize a collaborative network among the research teams responsible for protein mapping of individual chromosomes and to identify compelling biological and genetic mechanisms influencing colocated genes and their protein products. The C-HPP aims to foster the development of proteome analysis and integration of the findings from related molecular -omics technology platforms through collaborations among universities, industries, and private research groups. The C-HPP consortium leadership has elicited broad input for standard guidelines to manage these international efforts more efficiently by mobilizing existing resources and collaborative networks. The C-HPP guidelines set out the collaborative consensus of the C-HPP teams, introduce topics associated with experimental approaches, data production, quality control, treatment, and transparency of data, governance of the consortium, and collaborative benefits. A companion approach for the Biology and Disease-Driven HPP (B/D-HPP) component of the Human Proteome Project is currently being organized, building upon the Human Proteome Organizations organ-based and biofluid-based initiatives (www.hupo.org/research). The common application of these guidelines in the participating laboratories is expected to facilitate the goal of a comprehensive analysis of the human proteome.

A First Step Toward Completion of a Genome-Wide Characterization of the Human Proteome.

Almost 10 years after the human genome project (HGP) was completed in 2003, the characterization of proteins encoded by each gene located on a human chromosome remains elusive with respect to annotation, abundance, function, structural modification and post-transcriptionally modified isoforms. The Human Proteome Organization (HUPO) has recently established the Human Proteome Project (HPP) initiative under which the Chromosome-centric Human Proteome Project (C-HPP) and Biology/Disease-Driven HPP (B/DHPP) were placed. The C-HPP, a 10-year project with a finite end point (2012.9−2022.9), has progressed steadily since it was officially launched in Geneva in 2011 and following publication of a landmark paper. A general collaborative consensus has been built up and resulted in the C-HPP guidelines with description of defined goals that have been further consolidated through numerous workshops and conference calls during the past years, under the aegis of the HUPO HPP. The idea of documenting progress in C-HPP in a special issue of this journal was born during the Geneva HUPO congress at a meeting of the PIs of the chromosome teams with the goal of recording demonstrable progress by the initiative as well as outlining the planned impact of this project on biological research. This special issue will provide examples of the coming explosion of information in genomics, transcriptomics and proteomics. The papers published here contain a diverse set of topics including an industrial viewpoint, related technology development, examples of chromosome parts lists and databases with large scale protein identifications from samples of interest to the initiative, for example, placenta and liver. The goals of the C-HPP are to map and annotate the entire human protein set encoded in each chromosome through the cooperation of the 25-membered international consortium covering 24 chromosomes and mitochondria (see Figure 1). C-HPP can be categorized as dual types of projects: hypothesisdriven initiatives (seeking the biology, disease proteins and pathways) and a data gathering multinational project (constructing a knowledge base). For example, each chromosome group can select a chromosome based on specific disease interests (e.g., cancer, genetic disease) or targets (e.g., biomarker). The initiative does not represent any change in a typical proteomic experiment but rather that data sets are shared with all of the chromosome teams with a resulting growth in aggregated knowledge and more information on “missing or poorly characterized proteins”. We believe that the C-HPP initiative will support ongoing evolution in the proteomics field, such as the earlier adoption of information flowing from molecular biology advances, such as ENCODE, integrated transcriptomics/proteomic measurements, evolution in research organizations (from individual laboratories to international research alliances), deep proteomic discovery experiments, top-down analyses of protein variants generated from alternative splicing variants (ASVs) and alternative splicing transcripts (ASTs), the public deposition of data sets, better statistical tools for assessing extremely large data sets and the development of informatics systems and interfaces to allow the integration of genomic, proteomic and individual protein variation information.

Proteomic alterations of the variants of human aldehyde dehydrogenase isozymes correlate with hepatocellular carcinoma

To develop novel markers for hepatocellular carcinomas (HCC), 10 cases were analyzed by 2‐dimensional electrophoresis (2DE) and matrix‐assisted laser desorption ionization mass spectrometry. Results were compared to those of paired adjacent nontumorous liver tissues. Comparative analysis of the respective spot patterns in 2DE revealed that 3 variants of class 3 aldehyde dehydrogenase (ALDH‐3) newly appeared while 2 variants of ALDH‐2 diminished to undetectable levels in HCC. However, 4 ALDH‐1 variants with different pIs remained unaffected. This pattern of concomitant appearance and disappearance of ALDH‐3 and ALDH‐2 variants was consistently observed in all HCC tissues. Our results suggest that alterations of ALDH isozyme variants may be closely correlated to HCC and that proteomic analysis of these proteins might be a novel approach to identify the molecular events in detail during hepatocarcinogenesis.

Nictation, a dispersal behavior of the nematode Caenorhabditis elegans, is regulated by IL2 neurons

Many nematodes show a stage-specific behavior called nictation in which a worm stands on its tail and waves its head in three dimensions. Here we show that nictation is a dispersal behavior regulated by a specific set of neurons, the IL2 cells, in C. elegans. We established assays for nictation and showed that cholinergic transmission was required for nictation. Cell type–specific rescue experiments and genetic ablation experiments revealed that the IL2 ciliated head neurons were essential for nictation. Intact cilia in IL2 neurons, but not in other ciliated head neurons, were essential, as the restoration of the corresponding wild-type gene activity in IL2 neurons alone in cilia-defective mutants was sufficient to restore nictation. Optogenetic activation of IL2 neurons induced nictation, suggesting that signals from IL2 neurons are sufficient for nictation. Finally, we demonstrated that nictation is required for transmission of C. elegans to a new niche using flies as artificial carriers, suggesting a role of nictation as a dispersal and survival strategy under harsh conditions.

Role of Lung Apolipoprotein A-I in Idiopathic Pulmonary Fibrosis Antiinflammatory and Antifibrotic Effect on Experimental Lung Injury and Fibrosis

RATIONALE Idiopathic pulmonary fibrosis (IPF) is caused by alterations in expression of proteins involved in multiple pathways, including matrix deposition, inflammation, injury, and repair. OBJECTIVES To understand the pathogenic changes in lung protein expression in IPF and to evaluate apolipoprotein (Apo) A-I as a candidate therapeutic molecule. METHODS Two-dimensional electrophoresis was adopted for differential display proteomics. Reverse-transcriptase polymerase chain reaction, Western blotting, immunohistochemical staining, and ELISA were performed for identification and quantitative measurement of Apo A-I in bronchoalveolar lavage fluids from subjects with IPF and experimental bleomycin-induced mice. MEASUREMENTS AND MAIN RESULTS Sixteen protein spots showed differences in relative intensity between IPF (n = 14) and healthy control subjects (n = 8). Nano liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed increase of haptoglobulin and decrease of alpha(1)-antitrypsin, alpha(1)-antichymotrypsin, macrophage capping protein, angiotensinogen, hemoglobin chain B, Apo A-I, clusterin, protein disulfide isomerase A3, immunoglobulin, and complement C4A in IPF compared with normal control subjects (P = 0.006-0.044). Apo A-I concentrations were lower in bronchoalveolar lavage fluids from subjects with IPF (n = 28) than in normal control subjects (n = 18; P < 0.01). In bleomycin-treated mice, Apo A-I protein in BALF was lower than that in sham-treated control animals. Immunohistochemical analysis showed positive staining on intraalveolar macrophages and epithelial cells of the lungs. Intranasal treatment with Apo A-I protein reduced the bleomycin-induced increases in number of inflammatory cells and collagen deposition in sham-treated mice in a dose-dependent manner. CONCLUSIONS Alterations of several inflammatory and antiinflammatory proteins in the lungs may be related to the pathogenesis of IPF, and local treatment with Apo A-I is very effective against the development of experimental lung injury and fibrosis.