Cell cycle-specific UNG2 phosphorylations regulate protein turnover, activity and association with RPA

Human UNG2 is a multifunctional glycosylase that removes uracil near replication forks and in non-replicating DNA, and is important for affinity maturation of antibodies in B cells. How these diverse functions are regulated remains obscure. Here, we report three new phosphoforms of the non-catalytic domain that confer distinct functional properties to UNG2. These are apparently generated by cyclin-dependent kinases through stepwise phosphorylation of S23, T60 and S64 in the cell cycle. Phosphorylation of S23 in late G1/early S confers increased association with replication protein A (RPA) and replicating chromatin and markedly increases the catalytic turnover of UNG2. Conversely, progressive phosphorylation of T60 and S64 throughout S phase mediates reduced binding to RPA and flag UNG2 for breakdown in G2 by forming a cyclin E/c-myc-like phosphodegron. The enhanced catalytic turnover of UNG2 p-S23 likely optimises the protein to excise uracil along with rapidly moving replication forks. Our findings may aid further studies of how UNG2 initiates mutagenic rather than repair processing of activation-induced deaminase-generated uracil at Ig loci in B cells

Matrix metalloproteinase 2 and tissue inhibitor of matrix metalloproteinases 2 in the diagnosis of colorectal adenoma and cancer patients.

The aim of the study was to assess the importance of the measurement of matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of matrix metalloproteinases 2 (TIMP-2) in patients with colorectal cancer (CRC) in relation to clinicopathological features of tumor and patients' survival. Additionally, we determined serum MMP-2 and TIMP-2 in colorectal adenoma (CA) patients and healthy controls and compared them with tumor markers, CEA and CA 19-9. The serum levels of MMP-2 and TIMP-2 in 91 CRC patients, 28 CA subjects and 91 healthy controls were determined by ELISA method, but concentrations of CEA and CA 19-9 using MEIA method. Nonparametric statistical analyses were used. Serum levels of MMP-2 and TIMP-2 were significantly lower in CRC patients than in healthy subjects and decreased with tumor stage. Additionally, MMP-2 concentrations were significantly lower in patients with CRC than in CA group. Diagnostic sensitivity of TIMP-2 (59%) was the highest among biomarkers tested and increased in combined use with CEA (79%). Moreover, the area under ROC curve (AUC) of TIMP-2 was larger than AUC of MMP-2 in differentiation between CRC and healthy subjects, but lower than AUC of matrix metalloproteinase 2 in differentiation between colorectal cancer and adenoma. Our findings suggest clinical usefulness of TIMP-2 as a biomarker in the diagnosis of CRC, especially in combination with CEA. However, further investigation is necessary

Proteolysis in human breast and colorectal cancer

Proteolysis occurs when proteinase activity exceeds inhibitor activity. Proteolysis is normally tightly regulated and is involved in cancer invasion and metastasis. The aim of this study was to compare proteolysis in breast and colorectal cancer. Proteinase and inhibitor expression were analysed in paired tumour and normal tissue samples from 43 breast and 24 colorectal cancer patients using substrate zymography, Western blotting and quenched fluorescence substrate hydrolysis. The expression of the latent forms of matrix metalloproteinase-2 (MMP-2), MMP-3 and MMP-9, urokinase plasminogen activator (uPA), tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 expression were observed in both tumour and normal tissue samples from breast and colorectal tissue; however, expression was greater in the tumour tissue. Expression of active MMP-2 and MMP-9 and the total MMP activity were greater in tumour compared to normal samples in both tissues (P< 0.05). The expression of all proteinases and total MMP activity was greater in colorectal tissue than breast tissue samples. Breast and colorectal cancer demonstrated different proteinase profiles, however proteolysis in both tissues was greater in tumour tissue than normal tissue

Membrane-Associated Proteins of a Lipopolysaccharide-Deficient Mutant of Neisseria meningitidis Activate the Inflammatory Response through Toll-Like Receptor 2

The recent isolation of a lipopolysaccharide (LPS)-deficient mutant of Neisseria meningitidis has allowed us to explore the roles of other gram-negative cell wall components in the host response to infection. The experiments in this study were designed to examine the ability of this mutant strain to activate cells. Although it was clearly less potent than the parental strain, we found the LPS-deficient mutant to be a capable inducer of the inflammatory response in monocytic cells, inducing a response similar to that seen with Staphylococcus aureus. Cellular activation by the LPS mutant was related to expression of CD14, a high-affinity receptor for LPS and other microbial products, as well as Toll-like receptor 2, a member of the Toll family of receptors recently implicated in host responses to gram-positive bacteria. In contrast to the parental strain, the synthetic LPS antagonist E5564 did not inhibit the LPS-deficient mutant. We conclude that even in the absence of LPS, the gram-negative cell wall remains a potent inflammatory stimulant, utilizing signaling pathways independent of those involved in LPS signaling