RNA-binding protein MAC5A interacts with the 26S proteasome to regulate DNA damage response in Arabidopsis.

DNA damage response (DDR) in eukaryotes is essential for the maintenance of genome integrity in challenging environments. The regulatory mechanisms of DDR have been well-established in yeast and humans. However, increasing evidence supports the idea that plants seem to employ different signaling pathways that remain largely unknown. Here, we report the role of MODIFIER OF SNC1, 4-ASSOCIATED COMPLEX SUBUNIT 5A (MAC5A) in DDR in Arabidopsis (Arabidopsis thaliana). Lack of MAC5A in mac5a mutants causes hypersensitive phenotypes to methyl methanesulfonate (MMS), a DNA damage inducer. Consistent with this observation, MAC5A can regulate alternative splicing of DDR genes to maintain the proper response to genotoxic stress. Interestingly, MAC5A interacts with the 26S proteasome (26SP) and is required for its proteasome activity. MAC core subunits are also involved in MMS-induced DDR. Moreover, we find that MAC5A, the MAC core subunits, and 26SP may act collaboratively to mediate high-boron-induced growth repression through DDR. Collectively, our findings uncover the crucial role of MAC in MMS-induced DDR in orchestrating growth and stress adaptation in plants.

SMA1, a homolog of the splicing factor Prp28, has a multifaceted role in miRNA biogenesis in Arabidopsis.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that repress gene expression. In plants, the RNase III enzyme Dicer-like (DCL1) processes primary miRNAs (pri-miRNAs) into miRNAs. Here, we show that SMALL1 (SMA1), a homolog of the DEAD-box pre-mRNA splicing factor Prp28, plays essential roles in miRNA biogenesis in Arabidopsis. A hypomorphic sma1-1 mutation causes growth defects and reduces miRNA accumulation correlated with increased target transcript levels. SMA1 interacts with the DCL1 complex and positively influences pri-miRNA processing. Moreover, SMA1 binds the promoter region of genes encoding pri-miRNAs (MIRs) and is required for MIR transcription. Furthermore, SMA1 also enhances the abundance of the DCL1 protein levels through promoting the splicing of the DCL1 pre-mRNAs. Collectively, our data provide new insights into the function of SMA1/Prp28 in regulating miRNA abundance in plants.

Reagents for Isobaric Labeling Peptides in Quantitative Proteomics.

Currently, the commercial reagents for isobaric peptides labeling (TMT and iTRAQ) have some drawbacks, such as high cost in experiments, especially in quantitation for the modified peptides, and inconvenient handling for variable sizes of samples. Herein, we developed a set of 10-plex isobaric tags (IBT) with high stability and low cost. The labeled peptides were sensitively detected on Orbitrap Q Exactive MS with an MS2 resolution of 35 000 at 30% NCE, while the peptides were efficiently labeled over 97% by IBT at a ratio of 10:1 of reagent/peptide (w/w) in 200 mM TEAB buffer for 2 h. The IBT labeling was demonstrated with a wide dynamic range of 50-fold without obvious matrix effects on quantification. Importantly, there was little quantification bias found among the individual IBT tags, indicating that the peptides labeled by different tags were quantitatively comparable. The IBT 10-plex reagents were applied for dynamically monitoring the quantitative responses of phosphoproteome stimulated by EGF treatment in HeLa cells. In total, 5 361 unique phosphopeptides were identified, which reached a similar conclusion as others reported. The IBT reagents were therefore experimentally proven as a new type of reagents for isobaric peptides labeling and useful in a large quantity peptides of quantitative proteomics.

Peptide Biosynthesis with Stable Isotope Labeling from a Cell-free Expression System for Targeted Proteomics with Absolute Quantification.

Because of its specificity and sensitivity, targeted proteomics using mass spectrometry for multiple reaction monitoring is a powerful tool to detect and quantify pre-selected peptides from a complex background and facilitates the absolute quantification of peptides using isotope-labeled forms as internal standards. How to generate isotope-labeled peptides remains an urgent challenge for accurately quantitative targeted proteomics on a large scale. Herein, we propose that isotope-labeled peptides fused with a quantitative tag could be synthesized through an expression system in vitro, and the homemade peptides could be enriched by magnetic beads with tag-affinity and globally quantified based on the corresponding multiple reaction monitoring signals provided by the fused tag. An Escherichia coli cell-free protein expression system, protein synthesis using recombinant elements, was adopted for the synthesis of isotope-labeled peptides fused with Strep-tag. Through a series of optimizations, we enabled efficient expression of the labeled peptides such that, after Strep-Tactin affinity enrichment, the peptide yield was acceptable in scale for quantification, and the peptides could be completely digested by trypsin to release the Strep-tag for quantification. Moreover, these recombinant peptides could be employed in the same way as synthetic peptides for multiple reaction monitoring applications and are likely more economical and useful in a laboratory for the scale of targeted proteomics. As an application, we synthesized four isotope-labeled glutathione S-transferase (GST) peptides and added them to mouse sera pre-treated with GST affinity resin as internal standards. A quantitative assay of the synthesized GST peptides confirmed the absolute GST quantification in mouse sera to be measurable and reproducible.

The ERN1 transcription factor gene is a target of the CCaMK/CYCLOPS complex and controls rhizobial infection in Lotus japonicus.

Bacterial accommodation inside living plant cells is restricted to the nitrogen-fixing root nodule symbiosis. In many legumes, bacterial uptake is mediated via tubular structures called infection threads (ITs). To identify plant genes required for successful symbiotic infection, we screened an ethyl methanesulfonate mutagenized population of Lotus japonicus for mutants defective in IT formation and cloned the responsible gene, ERN1, encoding an AP2/ERF transcription factor. We performed phenotypic analysis of two independent L. japonicus mutant alleles and investigated the regulation of ERN1 via transactivation and DNA-protein interaction assays. In ern1 mutant roots, nodule primordia formed, but most remained uninfected and bacterial entry via ITs into the root epidermis was abolished. Infected cortical nodule cells contained bacteroids, but transcellular ITs were rarely observed. A subset exhibited localized cell wall degradation and loss of cell integrity associated with bacteroid spread into neighbouring cells and the apoplast. Functional promoter studies revealed that CYCLOPS binds in a sequence-specific manner to a motif within the ERN1 promoter and in combination with CCaMK positively regulates ERN1 transcription. We conclude that the activation of ERN1 by CCaMK/CYCLOPS complex is an important step controlling IT-mediated bacterial progression into plant cells.

The serum protein responses to treatment with Xiaoke Pill and Glibenclamide in type 2 diabetes patients.

AIM: The Xiaoke Pill containing Chinese herb extracts and Glibenclamide, is used in therapy for type 2 diabetes mellitus (T2DM), and is effective in reducing the risk of hypoglycemia and improving diabetes symptoms compared with Glibenclamide. We describe a quantitative proteomics project to measure the T2DM serum proteome response to the Xiaoke Pill and Glibenclamide. METHODS: Based on a recently conducted 48-week clinical trial comparing the safety and efficacy of Glibenclamide (n = 400) and Xiaoke Pill (n = 400), after matching for age, sex, BMI, drug dose and whether hypoglycemia occurred, 32 patients were selected for the serum based proteomic analysis and divided into four groups (with/without hypoglycemia treated with Xiaoke Pill or Glibenclamide, n = 8 for each group). We screened the differential serum proteins related to treatments and the onset of hypoglycemia using the iTRAQ labeling quantitative proteomics technique. Baseline and follow-up samples were used. RESULTS: The quantitative proteomics experiments demonstrated that 25 and 21 proteins differed upon treatment with the Xiaoke Pill in patients without and with hypoglycemia, respectively, while 24 and 25 proteins differed upon treatment with Glibenclamide in patients without and with hypoglycemia, respectively. The overlap of different proteins between the patients with and without hypoglycemia given the same drug treatment was much greater than between the patients given different drug treatments. CONCLUSIONS: We conclude that the serum proteins response to the two different anti-diabetic drug treatments may serve as a sensitive biomarker for evaluation of the therapeutic effects and continue investigations into the mechanism.

The survival mechanisms of thermophiles at high temperatures: an angle of omics.

Thermophiles are referred to as microorganisms with optimal growth temperatures of >60 °C. Over the past few years, a number of studies have been conducted regarding thermophiles, especially using the omics strategies. This review provides a systematic view of the survival physiology of thermophiles from an "omics" perspective, which suggests that the adaptive ability of thermophiles is based on a cooperative mode with multi-dimensional regulations integrating genomics, transcriptomics, and proteomics.

Assessing Transcription Regulatory Elements To Evaluate the Expression Status of Missing Protein Genes on Chromosomes 11 and 19.

During an investigation of missing proteins with the RNA-seq data acquired from three liver cancer cell lines, the majority of the missing protein coding genes (MPGs) located at chromosome 11 (chr11) had no corresponding mRNAs, while a high percentage of the MPGs on chr19 were detected at the mRNA level. The phenomenon, which was also observed in more than 40 cell lines, led to an inquiry of causation of the different transcriptional statuses of the MPGs in the two chromosomes. We hypothesized that the special chromatin structure was a key element to regulate MPG transcription. Upon a systematical comparison of the effects of DNase I hypersensitive sites (DHSs), transcription factors (TFs), and histone modifications toward these genes or MPGs with/without mRNA evidence in chr11 and 19, we attributed the poor transcription of the MPGs to the weak capacity of these transcription regulatory elements, regardless of which chromosome the MPGs were located. We further analyzed the gene contents in chr11 and found a number of genes related to sensory functions in the presence of chr11. We postulate that a high number of sensory-related genes, which are located within special chromatin structure, could bring a low detection rate of MPGs in chr11.

Biomarker Discovery and Verification of Esophageal Squamous Cell Carcinoma Using Integration of SWATH/MRM.

We propose an efficient integration of SWATH with MRM for biomarker discovery and verification when the corresponding ion library is well established. We strictly controlled the false positive rate associated with SWATH MS signals and carefully selected the target peptides coupled with SWATH and MRM. We collected 10 samples of esophageal squamous cell carcinoma (ESCC) tissues paired with tumors and adjacent regions and quantified 1758 unique proteins with FDR 1% at protein level using SWATH, in which 467 proteins were abundance-dependent with ESCC. After carefully evaluating the SWATH MS signals of the up-regulated proteins, we selected 120 proteins for MRM verification. MRM analysis of the pooled and individual esophageal tissues resulted in 116 proteins that exhibited similar abundance response modes to ESCC that were acquired with SWATH. Because the ESCC-related proteins consisted of a high percentile of secreted proteins, we conducted the MRM assay on patient sera that were collected from pre- and postoperation. Of the 116 target proteins, 42 were identified in the ESCC sera, including 11 with lowered abundances postoperation. Coupling SWATH and MRM is thus feasible and efficient for the discovery and verification of cancer-related protein biomarkers.

Insights from ENCODE on Missing Proteins: Why ß-Defensin Expression Is Scarcely Detected.

ß-Defensins (DEFBs) have a variety of functions. The majority of these proteins were not identified in a recent proteome survey. Neither protein detection nor the analysis of transcriptomic data based on RNA-seq data for three liver cancer cell lines identified any expression products. Extensive investigation into DEFB transcripts in over 70 cell lines offered similar results. This fact naturally begs the question­Why are DEFB genes scarcely expressed? After examining DEFB gene annotation and the physicochemical properties of its protein products, we postulated that regulatory elements could play a key role in the resultant poor transcription of DEFB genes. Four regions containing DEFB genes and six adjacent regions on chromosomes 6, 8, and 20 were carefully investigated using The Encyclopedia of DNA Elements (ENCODE) information, such as that of DNase I hypersensitive sites (DHSs), transcription factors (TFs), and histone modifications. The results revealed that the intensities of these ENCODE features were globally weaker than those in the adjacent regions. Impressively, DEFB-related regions on chromosomes 6 and 8 containing several non-DEFB genes had lower ENCODE feature intensities, indicating that the absence of DEFB mRNAs might not depend on the gene family but may be reliant upon gene location and chromatin structure.

Appraisal of the Missing Proteins Based on the mRNAs Bound to Ribosomes.

Considering the technical limitations of mass spectrometry in protein identification, the mRNAs bound to ribosomes (RNC-mRNA) are assumed to reflect the mRNAs participating in the translational process. The RNC-mRNA data are reasoned to be useful for appraising the missing proteins. A set of the multiomics data including free-mRNAs, RNC-mRNAs, and proteomes was acquired from three liver cancer cell lines. On the basis of the missing proteins in neXtProt (release 2014-09-19), the bioinformatics analysis was carried out in three phases: (1) finding how many neXtProt missing proteins have or do not have RNA-seq and/or MS/MS evidence, (2) analyzing specific physicochemical and biological properties of the missing proteins that lack both RNA-seq and MS/MS evidence, and (3) analyzing the combined properties of these missing proteins. Total of 1501 missing proteins were found by neither RNC-mRNA nor MS/MS in the three liver cancer cell lines. For these missing proteins, some are expected higher hydrophobicity, unsuitable detection, or sensory functions as properties at the protein level, while some are predicted to have nonexpressing chromatin structures on the corresponding gene level. With further integrated analysis, we could attribute 93% of them (1391/1501) to these causal factors, which result in the expression products scarcely detected by RNA-seq or MS/MS.

Tissue-Based Proteogenomics Reveals that Human Testis Endows Plentiful Missing Proteins.

Investigations of missing proteins (MPs) are being endorsed by many bioanalytical strategies. We proposed that proteogenomics of testis tissue was a feasible approach to identify more MPs because testis tissues have higher gene expression levels. Here we combined proteomics and transcriptomics to survey gene expression in human testis tissues from three post-mortem individuals. Proteins were extracted and separated with glycine- and tricine-SDS-PAGE. A total of 9597 protein groups were identified; of these, 166 protein groups were listed as MPs, including 138 groups (83.1%) with transcriptional evidence. A total of 2948 proteins are designated as MPs, and 5.6% of these were identified in this study. The high incidence of MPs in testis tissue indicates that this is a rich resource for MPs. Functional category analysis revealed that the biological processes that testis MPs are mainly involved in are sexual reproduction and spermatogenesis. Some of the MPs are potentially involved in tumorgenesis in other tissues. Therefore, this proteogenomics analysis of individual testis tissues provides convincing evidence of the discovery of MPs. All mass spectrometry data from this study have been deposited in the ProteomeXchange (data set identifier PXD002179).

Quest for Missing Proteins: Update 2015 on Chromosome-Centric Human Proteome Project.

This paper summarizes the recent activities of the Chromosome-Centric Human Proteome Project (C-HPP) consortium, which develops new technologies to identify yet-to-be annotated proteins (termed "missing proteins") in biological samples that lack sufficient experimental evidence at the protein level for confident protein identification. The C-HPP also aims to identify new protein forms that may be caused by genetic variability, post-translational modifications, and alternative splicing. Proteogenomic data integration forms the basis of the C-HPP's activities; therefore, we have summarized some of the key approaches and their roles in the project. We present new analytical technologies that improve the chemical space and lower detection limits coupled to bioinformatics tools and some publicly available resources that can be used to improve data analysis or support the development of analytical assays. Most of this paper's content has been compiled from posters, slides, and discussions presented in the series of C-HPP workshops held during 2014. All data (posters, presentations) used are available at the C-HPP Wiki (http://c-hpp.webhosting.rug.nl/) and in the Supporting Information.

sapFinder: an R/Bioconductor package for detection of variant peptides in shotgun proteomics experiments.

UNLABELLED: Single nucleotide variations (SNVs) located within a reading frame can result in single amino acid polymorphisms (SAPs), leading to alteration of the corresponding amino acid sequence as well as function of a protein. Accurate detection of SAPs is an important issue in proteomic analysis at the experimental and bioinformatic level. Herein, we present sapFinder, an R software package, for detection of the variant peptides based on tandem mass spectrometry (MS/MS)-based proteomics data. This package automates the construction of variation-associated databases from public SNV repositories or sample-specific next-generation sequencing (NGS) data and the identification of SAPs through database searching, post-processing and generation of HTML-based report with visualized interface. AVAILABILITY AND IMPLEMENTATION: sapFinder is implemented as a Bioconductor package in R. The package and the vignette can be downloaded at http://bioconductor.org/packages/devel/bioc/html/sapFinder.html and are provided under a GPL-2 license.

Quantitative proteomics reveals the temperature-dependent proteins encoded by a series of cluster genes in thermoanaerobacter tengcongensis.

Comprehensive and quantitative information of the thermophile proteome is an important source for understanding of the survival mechanism under high growth temperature. Thermoanaerobacter tengcongensis (T. tengcongensis), a typical anaerobic thermophilic eubacterium, was selected to quantitatively evaluate its protein abundance changes in response to four different temperatures. With optimized procedures of isobaric tags for relative and absolute quantitation quantitative proteomics (iTRAQ), such as peptide fractionation with high-pH reverse phase (RP) high performance liquid chromatography (HPLC), tandem MS acquisition mode in LTQ Orbitrap Velos MS, and evaluation of the quantification algorithms, high quality of the quantitative information of the peptides identified were acquired. In total, 1589 unique proteins were identified and defined 251 as the temperature-dependent proteins. Analysis of genomic locations toward the correspondent genes of these temperature-dependent proteins revealed that more than 30% were contiguous units with relevant biological functions, which are likely to form the operon structures in T. tengcongensis. The RNA sequencing (RNA-seq) data further demonstrated that these cluster genes were cotranscribed, and their mRNA abundance changes responding to temperature exhibited the similar trends as the proteomic results, suggesting that the temperature-dependent proteins are highly associated with the correspondent transcription status. Hence, the operon regulation is likely an energy-efficient mode for T. tengcongensis survival. In addition, evaluation to the functions of differential proteomes indicated that the abundance of the proteins participating in sulfur-respiration on the plasma membrane was decreased as the temperature increased, whereas the glycolysis-related protein abundance was increased. The energy supply in T. tengcongensis at high temperature is, therefore, speculated not mainly through the respiration chain reactions.

Comparison of the analgesic effects of cryoanalgesia vs. parecoxib for lung cancer patients after lobectomy.

This study was designed to compare the analgesic effects of cryoanalgesia and parecoxib in lung cancer patients after lobectomy. A total of 178 lung cancer patients awaiting large-sized lobectomy were enrolled in the study. The patients were randomly divided into Group A (intercostal nerve cryoanalgesia) and Group B (parecoxib). The analgesic and adverse effects were compared between the two groups. The pain score of Group A was significantly lower than that of Group B (P < 0.05). The patients in Group A used significantly less morphine than those in Group B (P < 0.05). There were also significantly fewer complications in Group A than in Group B (P < 0.05). Cryoanalgesia of the intercostal nerves can be considered an economical, safe and simple technique for the long-term management of post-lobectomy pain.

IQuant: an automated pipeline for quantitative proteomics based upon isobaric tags.

Quantitative proteomics technology based on isobaric tags is playing an important role in proteomic investigation. In this paper, we present an automated software, named IQuant, which integrates a postprocessing tool of protein identification and advanced statistical algorithms to process the MS/MS signals generated from the peptides labeled by isobaric tags and aims at proteomics quantification. The software of IQuant, which is freely downloaded at http://sourceforge.net/projects/iquant/, can run from a graphical user interface and a command-line interface, and can work on both Windows and Linux systems.

Omics evidence: single nucleotide variants transmissions on chromosome 20 in liver cancer cell lines.

Cancer genomics unveils many cancer-related mutations, including some chromosome 20 (Chr.20) genes. The mutated messages have been found in the corresponding mRNAs; however, whether they could be translated to proteins still requires more evidence. Herein, we proposed a transomics strategy to profile the expression status of human Chr.20 genes (555 in Ensembl v72). The data of transcriptome and translatome (the mRNAs bound with ribosome, translating mRNAs) revealed that ∼80% of the coding genes on Chr.20 were detected with mRNA signals in three liver cancer cell lines, whereas of the proteome identified, only ∼45% of the Chr.20 coding genes were detected. The high amount of overlapping of identified genes in mRNA and RNC-mRNA (ribosome nascent-chain complex-bound mRNAs, translating mRNAs) and the consistent distribution of the abundance averages of mRNA and RNC-mRNA along the Chr.20 subregions in three liver cancer cell lines indicate that the mRNA information is efficiently transmitted from transcriptional to translational stage, qualitatively and quantitatively. Of the 457 genes identified in mRNAs and RNC-mRNA, 136 were found to contain SNVs with 213 sites, and >40% of these SNVs existed only in metastatic cell lines, suggesting them as the metastasis-related SNVs. Proteomics analysis showed that 16 genes with 20 SNV sites were detected with reliable MS/MS signals, and some SNVs were further validated by the MRM approach. With the integration of the omics data at the three expression phases, therefore, we are able to achieve the overall view of the gene expression of Chr.20, which is constructive in understanding the potential trend of encoding genes in a cell line and exploration of a new type of markers related to cancers.

Chromosome-8-coded proteome of Chinese Chromosome Proteome Data set (CCPD) 2.0 with partial immunohistochemical verifications.

We upgraded the preliminary CCPD 1.0 to CCPD 2.0 using the latest deep-profiling proteome (CCPD 2013) of three hepatocellular carcinoma (HCC) cell lines, namely, Hep3B, MHCC97H, and HCCLM3 (ProteomeXchange identifiers: PXD000529, PXD000533, and PXD000535). CCPD 2.0 totally covered 63.6% (438/689) of Chr. 8-coded proteins and 62.6% (439/701) of Chr. 8-coded protein-coding genes. Interestingly, we found that the missing proteins exhibited a tendency to form a cluster region in chromosomes, such as two ß-defensins clusters in Chr. 8, caused perhaps by their inflammation-related features. For the 41 Chr. 8-coded proteins being weakly or barely identified previously, we have performed an immunohistochemical (IHC) verification in 30 pairs of carcinoma/para-carcinoma HCC and 20 noncancerous liver tissues and confirmed their expressional evidence and occurrence proportions in tissue samples. We also verified 13 Chr. 8-coded HCC tumorigenesis-associated depleting or deficient proteins reported in CCPD 1.0 using IHC and screened 16 positive and 24 negative HCC metastatic potential-correlated proteins from large-scale label-free proteome quantitation data of CCPD 2013. Our results suggest that the selection of proper samples and the methodology to look for targeted missing proteins should be carefully considered in further verifications for the remaining Chr. 8-coded proteins.

Systematic analyses of the transcriptome, translatome, and proteome provide a global view and potential strategy for the C-HPP.

To estimate the potential of the state-of-the-art proteomics technologies on full coverage of the encoding gene products, the Chinese Human Chromosome Proteome Consortium (CCPC) applied a multiomics strategy to systematically analyze the transciptome, translatome, and proteome of the same cultured hepatoma cells with varied metastatic potential qualitatively and quantitatively. The results provide a global view of gene expression profiles. The 9064 identified high confident proteins covered 50.2% of all gene products in the translatome. Those proteins with function of adhesion, development, reproduction, and so on are low abundant in transcriptome and translatome but absent in proteome. Taking the translatome as the background of protein expression, we found that the protein abundance plays a decisive role and hydrophobicity has a greater influence than molecular weight and isoelectric point on protein detectability. Thus, the enrichment strategy used for low-abundant transcription factors helped to identify missing proteins. In addition, those peptides with single amino acid polymorphisms played a significant role for the disease research, although they might negligibly contribute to new protein identification. The proteome raw and metadata of proteome were collected using the iProX submission system and submitted to ProteomeXchange (PXD000529, PXD000533, and PXD000535). All detailed information in this study can be accessed from the Chinese Chromosome-Centric Human Proteome Database.