Xinzhi Lu

GlcNAcylation Plays an Essential Role in Breast Cancer Metastasis

GlcNAcylation, a dynamic posttranslational modification, is involved in a wide range of biological processes and some human diseases. Although there is emerging evidence that some tumor-associated proteins are modified by GlcNAcylation, the role of GlcNAcylation in tumor progression remains unclear. Here, we show that GlcNAcylation enhances the migration/invasion of breast cancer cells in vitro and lung metastasis in vivo. The decrease of cell surface E-cadherin is the molecular mechanism underlying GlcNAcylation-induced breast cancer metastasis. p120 and beta-catenin, but not E-cadherin, are GlcNAcylated; the GlcNAcylation of p120 and beta-catenin might play roles in the decrease of cell surface E-cadherin. Moreover, immunohistochemistry analysis indicated that the global GlcNAcylation level in breast tumor tissues is elevated significantly as compared with that in the corresponding adjacent tissues; further, GlcNAcylation was significantly enhanced in metastatic lymph nodes compared with their corresponding primary tumor tissues. This is the first report to clearly elucidate the roles and mechanisms whereby GlcNAcylation influences the malignant properties of breast cancer cells. These results also suggest that GlcNAcylation might be a potential target for the diagnosis and therapy of breast cancer.

Molecular Cloning and Characterization of a Novel β-Agarase, AgaB, from Marine Pseudoalteromonas sp. CY24

Agarases are generally classified into glycoside hydrolase families 16, 50, and 86 and are found to degrade agarose to frequently generate neoagarobiose, neoagarotetraose, or neoagarohexaose as the main products. In this study we have cloned a novel endo-type β-agarase gene, agaB, from marine Pseudoalteromonas sp. CY24. The novel agarase encoded by agaB gene has no significant sequence similarity with any known proteins including all glycoside hydrolases. It degrades agarose to generate neoagarooctaose and neoagarodecaose as the main end products. Based on the analyses of enzymatic kinetics and degradation patterns of different oligosaccharides, the agarase AgaB appears to have a large substrate binding cleft that accommodates 12 sugar units, with 8 sugar units toward the reducing end spanning subsites +1 to +8 and 4 sugar units toward the non-reducing end spanning subsites -4 to -1, and enzymatic cleavage taking place between subsites -1 and +1. In addition, 1H NMR analysis shows that this enzyme hydrolyzes the glycosidic bond with inversion of anomeric configuration, in contrast to other known agarases that are retaining. Altogether, AgaB is structurally and functionally different from other known agarases and appears to represent a new family of glycoside hydrolase.

Silencing of GM3 synthase suppresses lung metastasis of murine breast cancer cells

BackgroundGangliosides are sialic acid containing glycosphingolipids that are ubiquitously distributed on vertebrate plasma membranes. GM3, a precursor for most of the more complex ganglioside species, is synthesized by GM3 synthase. Although total ganglioside levels are significantly higher in breast tumor tissue than in normal mammary tissue, the roles played by gangliosides in breast cancer formation and metastasis are not clear.MethodsTo investigate the roles of gangliosides in breast tumor development, GM3 synthase was silenced in the highly metastatic 4T1 cells and over-expressed in the non-metastatic 67NR cells. The behavior of breast cancer cells was examined in vitro using migration assay, invasion assay, and soft agar assay. Tumor formation and metastasis in vivo were examined using a well established mouse mammary tumor model.ResultsGM3 synthase silencing in 4T1 cells significantly inhibited cell migration, invasion and anchorage-independent growth in vitro, and lung metastasis in vivo. In addition, over-expression of GM3 synthase in nonmetastatic 67NR cells significantly induced cell migration and anchorage-independent growth. Further studies indicated that activation of the phosphoinositide-3 kinase/Akt pathway, and consequently inhibition of nuclear factor of activated T cell (NFAT)1 expression, could be the mechanism underlying the suppression of breast cancer migration/invasion induced by GM3 synthase silencing.ConclusionOur findings indicate that GM3 synthase silencing suppressed lung metastasis in murine breast cancer cells. The molecular mechanism that underlies GM3 synthase mediated migration and invasion was inhibition of the phosphoinositide-3 kinase/Akt pathway. The findings suggest that GM3 synthase may be of value as a therapeutic target in breast cancer.

Artificial trans-encoded small non-coding RNAs specifically silence the selected gene expression in bacteria

Recently, many small non-coding RNAs (sRNAs) with important regulatory roles have been identified in bacteria. As their eukaryotic counterparts, a major class of bacterial trans-encoded sRNAs acts by basepairing with target mRNAs, resulting in changes in translation and stability of the mRNA. RNA interference (RNAi) has become a powerful gene silencing tool in eukaryotes. However, such an effective RNA silencing tool remains to be developed for prokaryotes. In this study, we described first the use of artificial trans-encoded sRNAs (atsRNAs) for specific gene silencing in bacteria. Based on the common structural characteristics of natural sRNAs in Gram-negative bacteria, we developed the designing principle of atsRNA. Most of the atsRNAs effectively suppressed the expression of exogenous EGFP gene and endogenous uidA gene in Escherichia coli. Further studies demonstrated that the mRNA base pairing region and AU rich Hfq binding site were crucial for the activity of atsRNA. The atsRNA-mediated gene silencing was Hfq dependent. The atsRNAs led to gene silencing and RNase E dependent degradation of target mRNA. We also designed a series of atsRNAs which targeted the toxic genes in Staphyloccocus aureus, but found no significant interfering effect. We established an effective method for specific gene silencing in Gram-negative bacteria.

Cloning, expression and characterization of a new agarase-encoding gene from marine Pseudoalteromonas sp.

AbstractΤhe β-agarase gene agaA, cloned from a marine bacterium, Pseudoalteromonas sp. CY24, consists of 1,359 nucleotides encoding 453 amino acids in a sequence corresponding to a catalytic domain of glycosyl hydrolase family 16 (GH16) and a carbohydrate-binding module type 13 (CBM13). The recombinant enzyme is an endo-type agarase that hydrolyzes β-1,4-linkages of agarose, yielding neoagarotetraose and neoagarohexaose as the predominant products. In two cleavage patterns, AgaA digested the smallest substrate, neoagarooctaose, into neoagarobiose, neoagarotetraose and neoagarohexaose. Site directed mutation was performed to investigate the differences between AgaA and AgaD of Vibrio sp. PO-303, identifying residues V109VTS112 as playing a key role in the enzyme reaction.

O-GlcNAcylation of SIRT1 enhances its deacetylase activity and promotes cytoprotection under stress

SIRT1 is the most evolutionarily conserved mammalian sirtuin, and it plays a vital role in the regulation of metabolism, stress responses, genome stability, and ageing. As a stress sensor, SIRT1 deacetylase activity is significantly increased during stresses, but the molecular mechanisms are not yet fully clear. Here, we show that SIRT1 is dynamically modified with O-GlcNAc at Ser 549 in its carboxy-terminal region, which directly increases its deacetylase activity both in vitro and in vivo. The O-GlcNAcylation of SIRT1 is elevated during genotoxic, oxidative, and metabolic stress stimuli in cellular and mouse models, thereby increasing SIRT1 deacetylase activity and protecting cells from stress-induced apoptosis. Our findings demonstrate a new mechanism for the activation of SIRT1 under stress conditions and suggest a novel potential therapeutic target for preventing age-related diseases and extending healthspan.SIRT1 is a stress sensor whose deacetylase activity is increased during cellular stress, but the molecular mechanism is unclear. Here, the authors show that O-GlcNAcylation of SIRT1 is elevated upon different stress stimuli and increases SIRT1 deacetylase activity, protecting cells from stress-induced apoptosis.

Specific gene silencing by artificial trans-encoded small noncoding RNAs in bacteria

The authors of the paper ‘Specific gene silencing by artificial trans-encoded small noncoding RNAs in bacteria’ (Nucleic Acids Res. May 27, 2009. doi: 10.1093/nar/gkp447, PMID: 19474345 [Epub ahead of print]) wish it to be known that they have found several site mutations in the sequences of CY6 and N-CY1, which cause some changes in the secondary structure of the two atsRNAs. The conclusions drawn from the data of the two atsRNAs are therefore invalid. Accordingly, the authors are retracting the full article. The authors apologize for any inconvenience that this may have caused the scientific community and readers of the journal.