Glycine N‐methyltransferase (GNMT) is the main enzyme responsible for catabolism of excess hepatic S‐adenosylmethionine (SAMe). GNMT is absent in hepatocellular carcinoma (HCC), messenger RNA (mRNA) levels are significantly lower in livers of patients at risk of developing HCC, and GNMT has been proposed to be a tumor‐susceptibility gene for liver cancer. The identification of several children with liver disease as having mutations of the GNMT gene further suggests that this enzyme plays an important role in liver function. In the current study we studied development of liver pathologies including HCC in GNMT‐knockout (GNMT‐KO) mice. GNMT‐KO mice have elevated serum aminotransferase, methionine, and SAMe levels and develop liver steatosis, fibrosis, and HCC. We found that activation of the Ras and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways was increased in liver tumors from GNMT‐KO mice coincidently with the suppression of the Ras inhibitors Ras‐association domain family/tumor suppressor (RASSF) 1 and 4 and the JAK/STAT inhibitors suppressor of cytokine signaling (SOCS) 1–3 and cytokine‐inducible SH2‐protein. Finally, we found that methylation of RASSF1 and SOCS2 promoters and the binding of trimethylated lysine 27 in histone 3 to these 2 genes was increased in HCC from GNMT‐KO mice. Conclusion: These data demonstrate that loss of GNMT induces aberrant methylation of DNA and histones, resulting in epigenetic modulation of critical carcinogenic pathways in mice. (HEPATOLOGY 2008.)

Exosomes are extracellular vesicles of endosomal origin that are released by practically all cell types across metazoans. Exosomes are active vehicles of intercellular communication and can transfer lipids, RNAs, and proteins between different cells, tissues, or organs. Here, we describe a mechanism whereby proteins containing a KFERQ motif pentapeptide are loaded into a subpopulation of exosomes in a process that is dependent on the membrane protein LAMP2A. Moreover, we demonstrate that this mechanism is independent of the ESCRT machinery but dependent on HSC70, CD63, Alix, Syntenin-1, Rab31, and ceramides. We show that the master regulator of hypoxia HIF1A is loaded into exosomes by this mechanism to transport hypoxia signaling to normoxic cells. In addition, by tagging fluorescent proteins with KFERQ-like sequences, we were able to follow the interorgan transfer of exosomes. Our findings open new avenues for exosome engineering by allowing the loading of bioactive proteins by tagging them with KFERQ-like motifs.

Histone proteins are subject to a range of post-transcriptional modifications in living cells. The combinatorial nature of these modifications constitutes the “histone code” that dictates chromatin structure and function during development, growth, differentiation, and homeostasis of cells. Deciphering of the histone code is hampered by the lack of analytical methods for monitoring the combinatorial complexity of reversible multisite modifications of histones, including acetylation and methylation. To address this problem, we used LC-MSMS technology and Virtual Expert Mass Spectrometrist software for qualitative and quantitative proteomic analysis of histones extracted from human small cell lung cancer cells. A total of 32 acetylations, methylations, and ubiquitinations were located in the human histones H2A, H2B, H3, and H4, including seven novel modifications. An LC-MSMS-based method was applied in a quantitative proteomic study of the dose-response effect of the histone deacetylase inhibitor (HDACi) PXD101 on histone acetylation in human cell cultures. Triplicate LC-MSMS runs at six different HDACi concentrations demonstrated that PXD101 affects acetylation of histones H2A, H2B, H3, and H4 in a site-specific and dose-dependent manner. This unbiased analysis revealed that a relative increase in acetylated peptide from the histone variants H2A, H2B, and H4 was accompanied by a relative decrease of dimethylated Lys⁵⁷ from histone H2B. The dose-response results obtained by quantitative proteomics of histones from HDACi-treated cells were consistent with Western blot analysis of histone acetylation, cytotoxicity, and dose-dependent expression profiles of p21 and cyclin A2. This demonstrates that mass spectrometry-based quantitative proteomic analysis of post-translational modifications is a viable approach for functional analysis of candidate drugs, such as HDAC inhibitors.

Quantitative analysis of protein expression is an important tool for the examination of complex biological systems. Albeit its importance, quantitative proteomics is still a challenging task because of the high dynamic range of protein amounts in the cell and the variation in the physical properties of proteins. Stable isotope labeling by amino acids in cell culture (SILAC) has been successfully used in yeast and mammalian cells to measure relative protein abundance by mass spectrometry. Here we show for the first time that proteins from Arabidopsis thaliana cell cultures can be selectively isotope-labeled in vivo by growing cells in the presence of a single stable isotope-labeled amino acid. Among the tested amino acids ([²H₃]-leucine, [¹³C₆]arginine, and [²H₄]lysine), [¹³C₆]arginine proved to be the most suitable. Incorporation of [¹³C₆]arginine into the proteome was homogeneous and reached efficiencies of about 80%. [¹³C₆]Arginine-labeled A. thaliana suspension cells were used to study the regulation of glutathione S-transferase expression in response to abiotic stress caused by salicylic acid and to identify proteins that bind specifically to phosphorylated 14-3-3 binding motifs on synthesized bait peptides in affinity purification experiments. In conclusion, the combination of stable isotope labeling of plant cells and mass spectrometry is a powerful technology that can be applied to study complex biological processes that involve changes in protein expression such as cellular responses to various kinds of stress or activation of cell signaling.