Xingfeng Yin

As a well-known phenomenon, total mRNAs poorly correlate to proteins in their abundances as reported. Recent findings calculated with bivariate models suggested even poorer such correlation, whereas focusing on the translating mRNAs (ribosome nascent-chain complex-bound mRNAs, RNC-mRNAs) subset. In this study, we analysed the relative abundances of mRNAs, RNC-mRNAs and proteins on genome-wide scale, comparing human lung cancer A549 and H1299 cells with normal human bronchial epithelial (HBE) cells, respectively. As discovered, a strong correlation between RNC-mRNAs and proteins in their relative abundances could be established through a multivariate linear model by integrating the mRNA length as a key factor. The R(2) reached 0.94 and 0.97 in A549 versus HBE and H1299 versus HBE comparisons, respectively. This correlation highlighted that the mRNA length significantly contributes to the translational modulation, especially to the translational initiation, favoured by its correlation with the mRNA translation ratio (TR) as observed. We found TR is highly phenotype specific, which was substantiated by both pathway analysis and biased TRs of the splice variants of BDP1 gene, which is a key transcription factor of transfer RNAs. These findings revealed, for the first time, the intrinsic and genome-wide translation modulations at translatomic level in human cells at steady-state, which are tightly correlated to the protein abundance and functionally relevant to cellular phenotypes.

It has been a long debate whether the 98% 'non-coding' fraction of human genome can encode functional proteins besides short peptides. With full-length translating mRNA sequencing and ribosome profiling, we found that up to 3330 long non-coding RNAs (lncRNAs) were bound to ribosomes with active translation elongation. With shotgun proteomics, 308 lncRNA-encoded new proteins were detected. A total of 207 unique peptides of these new proteins were verified by multiple reaction monitoring (MRM) and/or parallel reaction monitoring (PRM); and 10 new proteins were verified by immunoblotting. We found that these new proteins deviated from the canonical proteins with various physical and chemical properties, and emerged mostly in primates during evolution. We further deduced the protein functions by the assays of translation efficiency, RNA folding and intracellular localizations. As the new protein UBAP1-AST6 is localized in the nucleoli and is preferentially expressed by lung cancer cell lines, we biologically verified that it has a function associated with cell proliferation. In sum, we experimentally evidenced a hidden human functional proteome encoded by purported lncRNAs, suggesting a resource for annotating new human proteins.

Recent phosphoproteomic characterizations of Bacillus subtilis, Escherichia coli, Lactococcus lactis, Pseudomonas putida, and Pseudomonas aeruginosa have suggested that protein phosphorylation on serine, threonine, and tyrosine residues is a major regulatory post-translational modification in bacteria. In this study, we carried out a global and site-specific phosphoproteomic analysis on the Gram-positive pathogenic bacterium Streptococcus pneumoniae. One hundred and two unique phosphopeptides and 163 phosphorylation sites with distributions of 47%/44%/9% for Ser/Thr/Tyr phosphorylations from 84 S. pneumoniae proteins were identified through the combined use of TiO(2) enrichment and LC-MS/MS determination. The identified phosphoproteins were found to be involved in various biological processes including carbon/protein/nucleotide metabolisms, cell cycle and division regulation. A striking characteristic of S. pneumoniae phosphoproteome is the large number of multiple species-specific phosphorylated sites, indicating that high level of protein phosphorylation may play important roles in regulating many metabolic pathways and bacterial virulence.

Protein modification by chemical reagents has played an essential role in the treatment of human diseases. However, the reagents currently used are limited to the covalent modification of cysteine and lysine residues. It is thus desirable to develop novel methods that can covalently modify other residues. Despite the fact that the carboxyl residues are crucial for maintaining the protein function, few selective labeling reactions are currently available. Here, we describe a novel reactive probe, 3-phenyl-2H-azirine, that enables chemoselective modification of carboxyl groups in proteins under both in vitro and in situ conditions with excellent efficiency. Furthermore, proteome-wide profiling of reactive carboxyl residues was performed with a quantitative chemoproteomic platform.

Abstract Genistein is a natural protein tyrosine kinase inhibitor that exerts anti‐cancer effect by inducing G2/M arrest and apoptosis. However, the phosphotyrosine signaling pathways mediated by genistein are largely unknown. In this study, we combined tyrosine phosphoprotein enrichment with MS‐based quantitative proteomics technology to globally identify genistein‐regulated tyrosine phosphoproteins aiming to depict genistein‐inhibited phosphotyrosine cascades. Our experiments resulted in the identification of 213 phosphotyrosine sites on 181 genistein‐regulated proteins. Many identified phosphoproteins, including nine protein kinases, eight receptors, five protein phosphatases, seven transcriptical regulators and four signal adaptors, were novel inhibitory effectors with no previously known function in the anti‐cancer mechanism of genistein. Functional analysis suggested that genistein‐regulated protein tyrosine phosphorylation mainly by inhibiting the activity of tyrosine kinase EGFR, PDGFR, insulin receptor, Abl, Fgr, Itk, Fyn and Src. Core signaling molecules inhibited by genistein can be functionally categorized into the canonial Receptor‐MAPK or Receptor‐PI3K/AKT cascades. The method used here may be suitable for the identification of inhibitory effectors and tyrosine kinases regulated by anti‐cancer drugs.