<i>Plasmodium falciparum</i> Sir2A Preferentially Hydrolyzes Medium and Long Chain Fatty Acyl Lysine

<i>Plasmodium falciparum</i> Sir2A (PfSir2A), a member of the sirtuin family of nicotinamide adenine dinucleotide-dependent deacetylases, has been shown to regulate the expression of surface antigens to evade the detection by host immune surveillance. It is thought that PfSir2A achieves this by deacetylating histones. However, the deacetylase activity of PfSir2A is weak. Here we present enzymology and structural evidence supporting that PfSir2A catalyzes the hydrolysis of medium and long chain fatty acyl groups from lysine residues more efficiently. Furthermore, <i>P. falciparum</i> proteins are found to contain such fatty acyl lysine modifications that can be removed by purified PfSir2A <i>in vitro</i>. Together, the data suggest that the physiological function of PfSir2A in antigen variation may be achieved by removing medium and long chain fatty acyl groups from protein lysine residues. The robust activity of PfSir2A would also facilitate the development of PfSir2A inhibitors, which may have therapeutic value in malaria treatment

Plasma miRNAs as Diagnostic and Prognostic Biomarkers for Ovarian Cancer

<div><p>Background</p><p>Most (70%) epithelial ovarian cancers (EOCs) are diagnosed late. Non-invasive biomarkers that facilitate disease detection and predict outcome are needed. The microRNAs (miRNAs) represent a new class of biomarkers. This study was to identify and validate plasma miRNAs as biomarkers in EOC.</p><p>Methodology/Principal Findings</p><p>We evaluated plasma samples of 360 EOC patients and 200 healthy controls from two institutions. All samples were grouped into screening, training and validation sets. We scanned the circulating plasma miRNAs by TaqMan low-density array in the screening set and identified/validated miRNA markers by real-time polymerase chain reaction assay in the training set. Receiver operating characteristic and logistic regression analyses established the diagnostic miRNA panel, which were confirmed in the validation sets. We found higher plasma miR-205 and lower let-7f expression in cases than in controls. MiR-205 and let-7f together provided high diagnostic accuracy for EOC, especially in patients with stage I disease. The combination of these two miRNAs and carbohydrate antigen-125 (CA-125) further improved the accuracy of detection. MiR-483-5p expression was elevated in stages III and IV compared with in stages I and II, which was consistent with its expression pattern in tumor tissues. Furthermore, lower levels of let-7f were predictive of poor prognosis in EOC patients.</p><p>Conclusions/Significance</p><p>Our findings indicate that plasma miR-205 and let-7f are biomarkers for ovarian cancer detection that complement CA-125; let-7f may be predictive of ovarian cancer prognosis.</p></div

Revealing CD38 Cellular Localization Using a Cell Permeable, Mechanism-Based Fluorescent Small-Molecule Probe

Nicotinamide adenine dinucleotide (NAD) is increasingly recognized as an important signaling molecule that affects numerous biological pathways. Thus, enzymes that metabolize NAD can have important biological functions. One NAD-metabolizing enzyme in mammals is CD38, a type II transmembrane protein that converts NAD primarily to adenosine diphosphate ribose (ADPR) and a small amount of cyclic adenosine diphosphate ribose (cADPR). Localization of CD38 was originally thought to be only on the plasma membrane, but later reports showed either significant or solely, intracellular CD38. With the efficient NAD-hydrolysis activity, the intracellular CD38 may lead to depletion of cellular NAD, thus producing harmful effects. Therefore, the intracellular localization of CD38 needs to be carefully validated. Here, we report the synthesis and application of a cell permeable, fluorescent small molecule (SR101–F-araNMN) that can covalently label enzymatically active CD38 with minimal perturbation of live cells. Using this fluorescent probe, we revealed that CD38 is predominately on the plasma membrane of Raji and retinoic acid (RA)-treated HL-60 cells. Additionally, the probe revealed no CD38 expression in K562 cells, which was previously reported to have solely intracellular CD38. The finding that very little intracellular CD38 exists in these cell lines suggests that the major enzymatic function of CD38 is to hydrolyze extracellular rather than intracellular NAD. The fluorescent activity-based probes that we developed allow the localization of CD38 in different cells to be determined, thus enabling a better understanding of the physiological function

Study design flowchart.

<p>We separated the samples into three sets: screening, training, and validation. The validation set included two phases to validate the best panel.</p

Pathological and demographic characteristics of EOC patients in the training and validation sets.

*<p>In phase II, some information was missing for Nanjing Center patients.</p

ROC of the panel (miR-205 and let-7f) for EOC cases.

<p>We established the miR-205 and let-7f panel in validation phase I and confirmed it in independent samples in phase II. In phase I, (A) the AUC was 0.831 (95% CI, 0.772 to 0.880; sensitivity = 62.4%, specificity = 92.9%) for cases; (B) 0.895 (95% CI, 0.822 to 0.967; sensitivity = 77.8%, specificity = 90.0%) for early-stage cases versus controls; and (C) 0.814 (95% CI, 0.700 to 0.929; sensitivity = 68.4%, specificity = 92.9%) for low CA-125 (<35 U/ml) cases versus controls. In phase II, (D) the AUC was 0.813 (95% CI, 0.759 to 0.860; sensitivity = 71.3%, specificity = 82.0%) for cases versus controls; (E) 0.783 (95% CI, 0.699 to 0.853; sensitivity = 70.0%, specificity = 82.0%) for early-stage cases versus controls; and (F) 0.726 (95% CI, 0.634 to 0.805; sensitivity = 64.3%, specificity = 72.0%) for low CA-125 (<35 U/ml) cases versus controls.</p

PFS by plasma let-7f level.

<p>(A) A PFS analysis for all patients (n = 360) revealed that lower plasma levels of let-7f were associated with poor prognosis (<i>P</i> = 0.006). Stage-specific Kaplan-Meier survival curves revealed that the <i>P</i> values of the log-rank test were 0.576 and 0.002 for stages I and II (B) and III and IV (C), respectively. The survival data were compared using the log-rank test, and let-7f expression levels in patients were defined as high or low relative to the median. NP, no progression; P, progression.</p

Cox regression analysis of EOC OS and PFS in relation to plasma let-7f levels.

<p>Cox regression analysis of EOC OS and PFS in relation to plasma let-7f levels.</p

The crystal structure of CD38 with cADPcR.

<p>(A) Structure of cADPcR complexed with wild-type CD38. The σA weighted Fo-Fc different electron density is shown as gray isomesh contoured at 2.5σ. H bonds are shown as dashed lines and colored in cyan (B) Binding comparison between <i>N</i>1-cIDPR (carbons in green) and cADPcR (carbons in cyan) complexed with CD38, showing overlap of their “northern” ribosyl phosphate motifs.</p

Highest scoring docked conformation of <i>N</i>1-cIDPR (A), 8-amino <i>N</i>1-cIDPR (B) and 8-(4-aminobutane)amino <i>N</i>1-cIDPR (C).

<p>The ligands are shown as sticks and the residues as lines. Color codes of the atoms of the ligand are C, cyan, O, red; N, dark blue and P, orange. Dashed black lines show the hydrogen bond interactions between the ligands and the enzyme. Hydrogen atoms are not shown for clarity.</p