Many non-histone proteins are lysine methylated and a novel function of this modification is to trigger the proteolysis of methylated proteins. Here, we report that the methylated lysine 142 of DNMT1, a major DNA methyltransferase that preserves epigenetic inheritance of DNA methylation patterns during DNA replication, is demethylated by LSD1. A novel methyl-binding protein, L3MBTL3, binds the K142-methylated DNMT1 and recruits a novel CRL4DCAF5 ubiquitin ligase to degrade DNMT1. Both LSD1 and PHF20L1 act primarily in S phase to prevent DNMT1 degradation by L3MBTL3-CRL4DCAF5. Mouse L3MBTL3/MBT-1 deletion causes accumulation of DNMT1 protein, increased genomic DNA methylation, and late embryonic lethality. DNMT1 contains a consensus methylation motif shared by many non-histone proteins including E2F1, a key transcription factor for S phase. We show that the methylation-dependent E2F1 degradation is also controlled by L3MBTL3-CRL4DCAF5. Our studies elucidate for the first time a novel mechanism by which the stability of many methylated non-histone proteins are regulated.

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DNA replication licensing occurs on chromatin, but how the chromatin template is regulated for replication remains mostly unclear. Here, we have analyzed the requirement of histone methyltransferases for a specific type of replication: the DNA re-replication induced by the downregulation of either Geminin, an inhibitor of replication licensing protein CDT1, or the CRL4CDT2 ubiquitin E3 ligase. We found that siRNA-mediated reduction of essential components of the MLL-WDR5-RBBP5 methyltransferase complexes including WDR5 or RBBP5, which transfer methyl groups to histone H3 at K4 (H3K4), suppressed DNA re-replication and chromosomal polyploidy. Reduction of WDR5/RBBP5 also prevented the activation of H2AX checkpoint caused by re-replication, but not by ultraviolet or X-ray irradiation; and the components of MLL complexes co-localized with the origin recognition complex (ORC) and MCM2-7 replicative helicase complexes at replication origins to control the levels of methylated H3K4. Downregulation of WDR5 or RBBP5 reduced the methylated H3K4 and suppressed the recruitment of MCM2-7 complexes onto replication origins. Our studies indicate that the MLL complexes and H3K4 methylation are required for DNA replication but not for DNA damage repair.

Glioma is one of the most common malignant tumor types of the central nervous system. It is necessary to identify biomarkers and novel therapeutic targets for glioma. The purpose of the present study was to distinguish lipid biomarkers with differential expression patterns in glioma tissues and normal brain tissues by matrix assisted laser desorption/ionization (MALDI)-imaging and MALDI-time of flight (TOF)-mass spectrometry (MS). Additionally, identification of lipid biomarkers was performed to describe novel therapeutic targets for glioma treatment. A total of six tissues from three patients with glioma and three control patients with traumatic brain injury were analyzed using UltrafleXtreme MALDI-TOF/TOF. The expression levels of 15 lipid peaks were higher in the TBT samples compared with in the GBT samples. The expression levels of another 16 lipid peaks were higher in the GBT samples compared with in the TBT samples. 14 peaks were identified as sphingomyelins using MS/MS. Additional results were also obtained from experiments using the glioma cell line U373-MG. These results indicated that treatment with the drug desipramine (Desi) inhibited the accumulation of ceramide on the cell membranes of glioma U373-MG cells. Treatment with Desi inhibited the activation of insulin-like growth factor-1 receptor and inhibited the activation of proteins in the PI3K/Akt signaling pathway.

In human, loss of acid sphingomyelinase (ASM/SMPD1) causes Niemann–Pick disease, type A. ASM hydrolyzes sphingomyelins to produce ceramides but protein targets of ASM remain largely unclear. Our mass spectrometry-based proteomic analyses have identified >100 proteins associated with the ASM-dependent, detergent-resistant membrane microdomains (lipid rafts), with >60% of these proteins being palmitoylated, including SNAP23, Src-family kinases Yes and Lyn, and Ras and Rab family small GTPases. Inactivation of ASM abolished the presence of these proteins in the plasma membrane, with many of them trapped in the Golgi. While palmitoylation inhibitors and palmitoylation mutants phenocopied the effects of ASM inactivation, we demonstrated that ASM is required for the transport of palmitoylated proteins, such as SNAP23 and Lyn, from the Golgi to the plasma membrane without affecting palmitoylation directly. Importantly, ASM delivered extracellularly can regulate the trafficking of SNAP23 from the Golgi to the plasma membrane. Our studies suggest that ASM, acting at the plasma membrane to produce ceramides, regulates the localization and trafficking of the palmitoylated proteins.

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AIM:To identify the protein expression differences related to the CagA-induced ERK pathway activation in AGS cells. METHODS:Human AGS cells transfected with cagA and blank vector were treated with specific mitogenactivated protein kinase kinase (MEK) inhibitor. Total cell proteins were combined by strong anion exchange (SAX2) and weak cation exchange (CM10) ProteinChip arrays and analyzed using surface-enhanced laser desorption/ ionization time-of-flight mass spectrometry (SELDI-TOF-MS) proteomics technology. Protein expression profiles were compared with those of inhibitor-untreated cagA transfectants. SwissProt/TrEMBL database searching for differentially expressed proteins was carried out using the TagIdent tool with the pI and mass information. RESULTS:When a total of 16 proteins that showed expression differences in inhibitor-untreated cagA transfectants were compared with vector transfectants, three proteins with m/z 4229, 8162 and 9084 were found to have no expression differences after treatment with MEK inhibitor, while the other 13 maintained the same expression differences after inhibitor treatment. Seven pieces of meaningful matching information for the three proteins were obtained from database searching. CONCLUSION:Biomarkers with m/z 4229, 8162 and 9084 are ERK1/2 phosphorylation dependent, and Ge Z et al . Biomarkers in CagA-affected AGS cells 555 www.wjgnet.com results in cell proliferation and differentiation [16] . As one of the major three members of the MAPKs, extracellular signal-regulated kinase (ERK) has been reported to be a key molecule in cell carcinogenesis in various cell types [17][18][19] . Higashi et al [20] have found that ERK is activated by CagA via SHP-2 recruitment and activation in AGS cells (human gastric adenocarcinoma epithelial cell line). Our previous research further proved that transformation of immortalized gastric epithelial cells by CagA takes place through the ERK/MAPK pathway [21] . However, the precise mechanism of the interactions between CagA and host cells in associated pathogenesis has not been fully elucidated.Recently, gene microarrays have been used to establish the global pattern of gene expression in cagA genetransfected AGS cells [22] . Nevertheless, gene and protein expression levels cannot easily be correlated or equated since proteins can exist in different posttranslational functional states. Therefore, it is important to gain an overall view of host response to CagA interference at the protein level. However, to date, few studies that exploit the global protein-expression pattern that can reflect host-cell response to CagA have been reported. Based on surface-enhanced laser desorption/ionization time-offlight mass spectrometry (SELDI-TOF-MS) technology, the ProteinChip platform has recently been shown to be valuable in establishing protein expression profiles and discovering new biomarkers [23][24][25] . ProteinChips, on which proteins from biological samples are selectively retained according to their biochemical properties, are analyzed ...