Proteogenomic Study beyond Chromosome 9: New Insight into Expressed Variant Proteome and Transcriptome in Human Lung Adenocarcinoma Tissues

This is a report of a human proteome project (HPP) related to chromosome 9 (Chr 9). To reveal missing proteins and undiscovered features in proteogenomes, both LC–MS/MS analysis and next-generation RNA sequencing (RNA-seq)-based identification and characterization were conducted on five pairs of lung adenocarcinoma tumors and adjacent nontumor tissues. Before our previous Chromosome-Centric Human Proteome Project (C-HPP) special issue, there were 170 remaining missing proteins on Chr 9 (neXtProt 2013.09.26 rel.); 133 remain at present (neXtProt 2015.04.28 rel.). In the proteomics study, we found two missing protein candidates that require follow-up work and one unrevealed protein across all chromosomes. RNA-seq analysis detected RNA expression for four nonsynonymous (NS) single nucleotide polymorphisms (SNPs) (in CDH17, HIST1H1T, SAPCD2, and ZNF695) and three synonymous SNPs (in CDH17, CST1, and HNF1A) in all five tumor tissues but not in any of the adjacent normal tissues. By constructing a cancer patient sample-specific protein database based on individual RNA-seq data and by searching the proteomics data from the same sample, we identified four missense mutations in four genes (LTF, HDLBP, TF, and HBD). Two of these mutations were found in tumor samples but not in paired normal tissues. In summary, our proteogenomic study of human primary lung tumor tissues detected additional and revealed novel missense mutations and synonymous SNP signatures, some of which are specific to lung cancers. Data from mass spectrometry have been deposited in the ProteomeXchange with the identifier PXD002523

N-Terminal Truncated UCH-L1 Prevents Parkinson's Disease Associated Damage

<div><p>Ubiquitin C-terminal hydrolase-L1 (UCH-L1) has been proposed as one of the Parkinson's disease (PD) related genes, but the possible molecular connection between UCH-L1 and PD is not well understood. In this study, we discovered an N-terminal 11 amino acid truncated variant UCH-L1 that we called NT-UCH-L1, in mouse brain tissue as well as in NCI-H157 lung cancer and SH-SY5Y neuroblastoma cell lines. <i>In vivo</i> experiments and hydrogen-deuterium exchange (HDX) with tandem mass spectrometry (MS) studies showed that NT-UCH-L1 is readily aggregated and degraded, and has more flexible structure than UCH-L1. Post-translational modifications including monoubiquitination and disulfide crosslinking regulate the stability and cellular localization of NT-UCH-L1, as confirmed by mutational and proteomic studies. Stable expression of NT-UCH-L1 decreases cellular ROS levels and protects cells from H₂O₂, rotenone and CCCP-induced cell death. NT-UCH-L1-expressing transgenic mice are less susceptible to degeneration of nigrostriatal dopaminergic neurons seen in the MPTP mouse model of PD, in comparison to control animals. These results suggest that NT-UCH-L1 may have the potential to prevent neural damage in diseases like PD.</p></div

Generation of hNT-UCH-L1-myc transgenic mice and the protective role of NT-UCH-L1 in the MPTP model of Parkinson's disease.

<p>(A) NT-UCH-L1 was constructed under control of the CAG promoter. (B) Quantification of the mRNAs of the NT-UCH-L1-myc transgene from five transgenic mice lines using quantitative RT-PCR. (C) Immunoprecipitation analysis of hNT-UCH-L1-myc in control and NT-Tg mouse brain using anti-myc antibody. Immunoprecipitants were analyzed by immunoblotting method using anti-myc antibody. 3^rd land, cell lysate of HEK293 cells transiently expressing NT-UCH-L1-myc. (D) Immunohistochemical analysis showing neuroprotective effect of NT-UCH-L1 on nigrostriatal dopaminergic neurons. Mice in each group (non-Tg control (a, b), non-Tg MPTP (c, d), NT-UCH-L1 control (e, f) and NT-UCH-L1 MPTP (g, h)) were sacrificed 7 d after the last MPTP injection (c, d, g, h) or PBS as controls (a, b, e, f) and brain tissues were processed for tyrosine hydroxylase (TH) immunostaining in the substantia nigra pars compacta (SN, a, c, e, g) and striatum (STR, b, d, f, h). Scale bar, a, c, e, g, 100((m; b, d, f, h, 50((m. (E, F) The number of TH-positive cells in the SN (D) and optical density of TH-positive striatal fibers (D) are shown in graphs. n = 3 or 4 for each experimental group.</p

Discovery of NT-UCH-L1 in human lung cancer and neuroblastoma cell lines and its enzymatic and structural differences from UCH-L1.

<p>(A) NCI-H157 (left panels) and SH-SY5Y (right panels) cells were analyzed using 2D-PAGE and visualized by Western blot analysis using anti-UCH-L1 (top panels), anti-N-terminal peptide (against the peptide, ¹MQLKPMEINPE¹¹) antibodies (lower panels), or silver staining (middle panels). Four spots representing UCH-L1 are numbered from 1 to 4. (B) Mouse whole brain tissue was analyzed using 2D-PAGE and visualized by Western blot analysis using anti-UCH-L1 (top panel) and anti-N-terminal peptide antibodies (lower panel). The location of NT was marked by a circle in each panel. (C) A diagram of UCH-L1 and N-terminal 11 amino acid truncated NT-UCH-L1. (D) Ubiquitin hydrolase activities of GST-UCH-L1 and GST-NT-UCH-L1 were measured using Ub-AMC as a substrate. 5 nM of GST-UCH-L1 or GST-NT-UCH-L1 was incubated with 0–1000 nM of Ub-AMC and monitored the release of free AMC at 460 nm. (E) HeLa cells transiently expressing NT-UCH-L1 were subjected to migration assay using transwell coated with Matrigel™. After 24 h of seeding, the number of migrated cells in the lower chamber was counted. The expression of NT-UCH-L1 in HeLa cells were verified by Western blot analysis using anti-myc antibody, and anti-α-tubulin antibody as a loading control.</p

Localization of NT-UCH-L1 in mitochondria.

<p>(A) HeLa cells stably expressing UCH-L1-myc, NT-UCH-L1-myc and K15/157R NT-UCH-L1-myc were stained with anti-myc and Alexa Fluor 488 secondary antibody (green). Mitochondria and nucleus were stained with Mitotracker (red) and DAPI (blue), respectively. Cells were visualized by confocal microscopy. (B) Cells were fractionated into cytosolic and mitochondrial fractionations. UCH-L1s were immunoblotted using anti-myc antibody and Hsp60 and PRX6, a mitochondrial and cytosolic marker protein, respectively, were immunoblotted using anti-Hsp60 and anti-PRX6 antibodies. The relative distribution of UCH-L1-myc, NT-UCH-L1-myc and K15/157R NT-UCH-L1-myc in the cytosolic and mitochondrial fractions were quantified by measuring their density (C). The mean ± s.d. of three independent experiments is shown. (D) Mitochondrial fractions of HeLa cells expressing NT-UCH-L1 were incubated with various concentrations of proteinase K for 1 h at 50°C and immunoblotted with anti-myc, Tom20, cytochrome C and COX4 antibodies. (E) Cells were treated 10((M CCCP for 0, 1, or 6 h and fractionated into cytosolic and mitochondrial fractionations. UCH-L1s were immunoblotted using anti-myc antibody and Hsp60, a mitochondrial marker protein, was immunoblotted using anti-Hsp60 antibody. (F) Cells were treated with 10((M CCCP in combination with 10((M MG132 (an inhibitor of proteasomal degradation), 10 mM NH4Cl (an inhibitor of lysosomal degradation) or 10 mM 3MA (an inhibitor of autophagy). UCH-L1s were immunoblotted using anti-myc antibody.</p

Degradation and aggregation-proneness of NT-UCH-L1.

<p>HeLa cells stably expressing UCH-L1-myc, NT-UCH-L1-myc and K15/157R NT-UCH-L1-myc were examined. (A, B) Cells were treated with 10 µg/mL cycloheximide for the indicated times and immunoblotted using anti-myc antibody (A) and the bands were quantified (B). (C, D) Cells were treated with DMSO, 10 µM MG132, 10 mM NH₄Cl or 10 mM 3MA 16 h and immunoblotted using anti-myc antibody (C) and the bands were quantified (D). (E, F) Cells were treated with DMSO or 10 µM MG132 for 9 h and divided into soluble and insoluble fractions. Samples were immunoblotted using anti-myc antibody (E) and the bands were quantified (F). Quantitative analysis was done and the mean ± s.d. of three independent experiments is shown. (G) SN4741 cells were transiently transfected with pcDNA3.1 UCH-L1-myc, pcDNA3.1 NT-UCH-L1-myc, and pcDNA3.1 K15/157R NT-UCH-L1-myc and fractionated into soluble and insoluble fractions. Fractions were immunoblotted using anti-myc and anti-α-tubulin. Due to the higher expression level of UCH-L1 than NT-UCH-L1s, immunoblot was done in separate membrane and the border lines were drawn. (H) SN4741 cells were transiently transfected with various amounts of pcDNA3.1 NT-UCH-L1-myc expressing plasmid. Cells were divided into soluble and insoluble fractions and analyzed in non-reducing ((-) 2-ME) and reducing ((+) 2-ME) gels. Proteins were immunoblotted using anti-myc and anti-α-tubulin antibodies. (I) HeLa stable cells expressing UCH-L1-myc and NT-UCH-L1-myc were treated with DMSO or 10 µM MG132 for 16 h. Myc tagged proteins were probed with anti-myc and Alexa Flour 488 secondary antibody (green), mitochondria were stained with Mitotracker (red) and nucleus were stained with DAPI (blue). Cells were examined under confocal microscope.</p

Structural differences between UCH-L1 and NT-UCH-L1.

<p>(A, B) Purified UCH-L1 and NT-UCH-L1 were subjected to HDX studies. UCH-L1 and NT-UCH-L1 were incubated with D₂O exchange buffer at 25°C for the indicated times and analyzed using nanoAcquity™/ESI/MS. HDX spectra of UCH-L1 and NT-UCH-L1 and deuterium exchange rates were represented by % exchange (A) and mass increase (B). (C) Recombinant UCH-L1 and NT-UCH-L1 were subjected to HDX studies. Proteins were incubated with D₂O exchange buffer at 25°C for 30 min, digested with trypsin and analyzed using nanoAcquity™/ESI/MS. Deuterium exchange rates were represented by % exchange and colored accordingly.</p

Protective effect of NT-UCH-L1 on PD related toxin induced damage.

<p>HeLa cells stably expressing UCH-L1-myc and NT-UCH-L1-myc were treated with 2 mM H₂O₂ for 1 h before plating cells (A) or 500 nM rotenone (B) and 25 µM CCCP (C) from the time indicated by arrow to the end of the experiment. Cell growth was monitored using real-time cell analyzer. Values represent mean of triplicates ± s.d. Black, mock; blue, UCH-L1; magenta, NT-UCH-L1.</p

Disulfide bonds identified in aggregated NT-UCH-L1 by nanoUPLC-ESI-q-TOF tandem MS combining DBond searching algorithm.

<p>Disulfide bonds identified in aggregated NT-UCH-L1 by nanoUPLC-ESI-q-TOF tandem MS combining DBond searching algorithm.</p

A suggested mechanism for molecular regulation of NT-UCH-L1.

<p>NT, NT-UCH-L1; Ub, monoubiquitin; two red circles, disulfide bond.</p