Sequential posttranslational modifications program FEN1 degradation during cell-cycle progression.

We propose that cell-cycle-dependent timing of FEN1 nuclease activity is essential for cell-cycle progression and the maintenance of genome stability. After DNA replication is complete at the exit point of the S phase, removal of excess FEN1 may be crucial. Here, we report a mechanism that controls the programmed degradation of FEN1 via a sequential cascade of posttranslational modifications. We found that FEN1 phosphorylation stimulated its SUMOylation, which in turn stimulated its ubiquitination and ultimately led to its degradation via the proteasome pathway. Mutations or inhibitors that blocked the modification at any step in this pathway suppressed FEN1 degradation. Critically, the presence of SUMOylation- or ubiquitination-defective, nondegradable FEN1 mutant protein caused accumulation of Cyclin B, delays in the G1 and G2/M phases, and polyploidy. These findings may represent a newly identified regulatory mechanism used by cells to ensure precise cell-cycle progression and to prevent transformation.

Discovery and Validation of Salivary Extracellular RNA Biomarkers for Noninvasive Detection of Gastric Cancer.

BACKGROUND: Biomarkers are needed for noninvasive early detection of gastric cancer (GC). We investigated salivary extracellular RNA (exRNA) biomarkers as potential clinical evaluation tools for GC. METHODS: Unstimulated whole saliva samples were prospectively collected from 294 individuals (163 GC and 131 non-GC patients) who underwent endoscopic evaluation at the Samsung Medical Center in Korea. Salivary transcriptomes of 63 GC and 31 non-GC patients were profiled, and mRNA biomarker candidates were verified with reverse transcription quantitative real-time PCR (RT-qPCR). In parallel, microRNA (miRNA) biomarkers were profiled and verified with saliva samples from 10 GC and 10 non-GC patients. Candidate biomarkers were validated with RT-qPCR in an independent cohort of 100/100 saliva samples from GC and non-GC patients. Validated individual markers were configured into a best performance panel. RESULTS: We identified 30 mRNA and 15 miRNA candidates whose expression pattern associated with the presence of GC. Among them, 12 mRNA and 6 miRNA candidates were verified with the discovery cohort by RT-qPCR and further validated with the independent cohort (n = 200). The configured biomarker panel consisted of 3 mRNAs (SPINK7, PPL, and SEMA4B) and 2 miRNAs (MIR140-5p and MIR301a), which were all significantly down-regulated in the GC group, and yielded an area under the ROC curve (AUC) of 0.81 (95% CI, 0.72-0.89). When combined with demographic factors, the AUC of the biomarker panel reached 0.87 (95% CI, 0.80-0.93). CONCLUSIONS: We have discovered and validated a panel of salivary exRNA biomarkers with credible clinical performance for the detection of GC. Our study demonstrates the potential utility of salivary exRNA biomarkers in screening and risk assessment for GC.

Elucidation of binding sites of dual antagonists in the human chemokine receptors CCR2 and CCR5.

Design of dual antagonists for the chemokine receptors CCR2 and CCR5 will be greatly facilitated by knowledge of the structural differences of their binding sites. Thus, we computationally predicted the binding site of the dual CCR2/CCR5 antagonist N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-benzohepten-8-yl] carbonyl]amino]benzyl]tetrahydro-2H-pyran-4-aminium (TAK-779), and a CCR2-specific antagonist N-(carbamoylmethyl)-3-trifluoromethyl benzamido-parachlorobenzyl 3-aminopyrrolidine (Teijin compound 1) in an ensemble of predicted structures of human CCR2 and CCR5. Based on our predictions of the protein-ligand interactions, we examined the activity of the antagonists for cells expressing thirteen mutants of CCR2 and five mutants of CCR5. The results show that residues Trp98(2.60) and Thr292(7.40) contribute significantly to the efficacy of both TAK-779 and Teijin compound 1, whereas His121(3.33) and Ile263(6.55) contribute significantly only to the antagonistic effect of Teijin compound 1 at CCR2. Mutation of residues Trp86(2.60) and Tyr108(3.32) adversely affected the efficacy of TAK-779 in antagonizing CCR5-mediated chemotaxis. Y49A(1.39) and E291A(7.39) mutants of CCR2 showed a complete loss of CCL2 binding and chemotaxis, despite robust cell surface expression, suggesting that these residues are critical in maintaining the correct receptor architecture. Modeling studies support the hypothesis that the residues Tyr49(1.39), Trp98(2.60), Tyr120(3.32), and Glu291(7.39) of CCR2 form a tight network of aromatic cluster and polar contacts between transmembrane helices 1, 2, 3, and 7.

Elucidating the Conformational Dependence of Calculated pKa Values.

The variability within calculated protein residue pKa values calculated using Poisson-Boltzmann continuum theory with respect to small conformational fluctuations is investigated. As a general rule, sites buried in the protein core have the largest pKa fluctuations but the least amount of conformational variability; conversely, sites on the protein surface generally have large conformational fluctuations but very small pKa fluctuations. These results occur because of the heterogeneous or uniform nature of the electrostatic microenvironments at the protein core or surface, respectively. Atypical surface sites with large pKa fluctuations occur at the interfaces between significant anionic and cationic potentials.