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.
Background: The prognosis of esophageal squamous cell carcinoma (ESCC) is generally poor, and the identification of molecular markers related to the regulation of ESCC invasion and migration is important. Methods and Results: In this study, we report that ring finger protein-128 (RNF128) enhances the invasiveness and motility of ESCC cells by using transwell assays and Western blotting. A xenograft nude mouse model showed that RNF128 promotes the metastasis of ESCC cells in the lung. A signal pathway analysis identified the epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK)/matrix matalloproteinases 2 (MMP-2) cascade as a mediator of RNF128-induced enhancement of ESCC progression. Inhibition experiments using inhibitors of EGFR, ERK kinase (MEK)/extracellular-signal-regulated-kinase (ERK), and MMP-2 reversed this progression. Co-immunoprecipitation demonstrated that RNF128 promotes the activation of the EGFR/ERK/MMP-2 pathway by interacting with p53 and p53 interacting with EGFR. Conclusion: Our results establish the functional role of RNF128 in driving the invasion and metastasis of ESCC through the EGFR/MAPK/MMP-2 pathway, implicating its potential as a candidate therapeutic target and prognostic biomarker for ESCC.
Exosomes are deliverers of critically functional proteins, capable of transforming target cells in numerous cancers, including hepatocellular carcinoma (HCC). We hypothesize that the motility of HCC cells can be featured by comparative proteome of exosomes. Hence, we performed the super-SILAC-based MS analysis on the exosomes secreted by three human HCC cell lines, including the non-motile Hep3B cell, and the motile 97H and LM3 cells. More than 1400 exosomal proteins were confidently quantified in each MS analysis with highly biological reproducibility. We justified that 469 and 443 exosomal proteins represented differentially expressed proteins (DEPs) in the 97H/Hep3B and LM3/Hep3B comparisons, respectively. These DEPs focused on sugar metabolism-centric canonical pathways per ingenuity pathway analysis, which was consistent with the gene ontology analysis on biological process enrichment. These pathways included glycolysis I, gluconeogenesis I and pentose phosphate pathways; and the DEPs enriched in these pathways could form a tightly connected network. By analyzing the relative abundance of proteins and translating mRNAs, we found significantly positive correlation between exosomes and cells. The involved exosomal proteins were again focusing on sugar metabolism. In conclusion, motile HCC cells tend to preferentially export more sugar metabolism-associated proteins via exosomes that differentiate them from non-motile HCC cells.
The biological function of many mitochondrial proteins in mechanistic detail has not been well investigated in clear cell renal cell carcinoma (ccRCC). A seven‐mitochondrial‐gene signature was generated by Lasso regression analysis to improve the prediction of prognosis of patients with ccRCC, using The Cancer Genome Atlas and Clinical Proteomic Tumor Analysis Consortium cohort. Among those seven genes, EFHD1 is less studied and its role in the progression of ccRCC remains unknown. The decreased expression of EFHD1 was validated in clinical samples and was correlated with unfavorable outcome. Overexpression of EFHD1 in ccRCC cells resulted in the reduction of mitochondrial Ca 2+ , and the inhibition of cell migration and invasion in vitro and tumor metastasis in vivo. Mechanistically, EFHD1 physically bound to the core mitochondrial calcium transporter (mitochondrial calcium uniporter, MCU) through its N‐terminal domain. The interaction between EFHD1 and MCU suppressed the uptake of Ca 2+ into mitochondria, and deactivated the Hippo/YAP signaling pathway. Further data revealed that the ectopic expression of EFHD1 upregulated STARD13 to enhance the phosphorylation of YAP protein at Ser‐127. The knockdown of STARD13 or the overexpression of MCU partly abrogated the EFHD1‐mediated induction of phosphorylation of YAP at Ser‐127 and suppression of cell migration. Taken together, the newly identified EFHD1–MCU–STARD13 axis participates in the modulation of the Hippo/YAP pathway and serves as a novel regulator in the progression of ccRCC.
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