CTCF Mediates Replicative Senescence Through POLD1

POLD1, the catalytic subunit of DNA polymerase δ, plays a critical role in DNA synthesis and DNA repair processes. Moreover, POLD1 is downregulated in replicative senescence to mediate aging. In any case, the components of age-related downregulation of POLD1 expression have not been fully explained. In this article, we elucidate the mechanism of the regulation of POLD1 at the transcription level and found that the transcription factor CCCTC-binding factor (CTCF) was bound to the POLD1 promoter area in two sites. The binding level of CTCF for the POLD1 promoter appeared to be related to aging and was confirmed to be positively controlled by the CTCF level. Additionally, cell senescence characteristics were detected within the cells transfected with short hairpin RNA (shRNA)-CTCF, pLenti-CMV-CTCF, shRNA-POLD1, and pLenti-CMV-POLD1, and the results showed that the CTCF may contribute to the altered expression of POLD1 in aging. In conclusion, the binding level of CTCF for the POLD1 promoter intervened by an age-related decrease in CTCF and downregulated the POLD1 expression in aging. Moreover, the decrease in CTCF-mediated POLD1 transcription accelerates the progression of cell aging.

P0866EVALUATION OF ERYPTOSIS IN PATIENTS WITH CHRONIC KIDNEY DISEASE

Background and Aims One of the many factors in the pathogenesis of renal anaemia, is eryptosis. The aim of this study is to evaluate eryptosis in patients with chronic renal disease stage 3-5 and 5 dialysis (5D). Method Patients with CKD who were monitored in our nephrology department were included in the study. The age range of the patient groups was 18-80 years and haemoglobin levels were <13 g / dl in men and <12 g / dl in women. As parameters of eryptosis, flow cytometric annexin V binding and intracellular calcium, and superoxide dismutase (SOD) activity with ELISA method were measured in 59 predialysis patient (eGFR<60 mL / min 1.73 m2), 26 haemodialysis patient, 21 peritoneal dialysis patient and 29 healthy volunteers with similar age (control). Haematological and biochemical tests including complete blood count(CBC), erythrocyte sedimentation rate (ESR), Parathormone (PTH), 25-OH vit D3, Blood urea nitrogen (BUN), Creatinine (Cr), C-reactive protein (CRP), Na, K, phosphor (P), ionized and total Calcium, Ferritin, B12 and Folate, were performed. IBM SPSS Statistics Version 20.0 was used for statistical analysis of the data Results The amount of intracellular calcium were 3.05 ± 1.66 in predialysis group, 2.24 ± 0.99 in haemodialysis group, 2.38 ± 0.87 in peritoneal dialysis group and 1.71 ± 0.46 in control group. For intracellular calcium there was statistically significant difference only between predialysis group and control group (p <0. 000). Annexin V binding were calculated as 1.05 ± 0.99 in predialysis group, 1.15 ± 0.56 in haemodialysis group, 1.06 ± 0.87 in peritoneal dialysis group and 0.88 ± 0.86 in control group. Comparing to control group annexin V binding in predialysis, haemodialysis and peritoneal dialysis groups, were statistically significantly increased (for all p <0.000). SOD activity was 0.07 ± 0.07 in predialysis group, 0.13 ± 0.08 in haemodialysis group, 0.14 ± 0.07 in peritoneal dialysis group and 0.03 ± 0.01 in control group. The difference between the control group and dialysis group was statistically significant (p <0. 00). Comparing to nonusers; patients using calcium channel blockers (CCB) had low annexin V binding level (p = 0.013). Patients using erythropoietin (EPO) had elevated annexin V binding level (p <0.000) and lower intracellular calcium (p = 0.014) according to non- users. Conclusion We determined increased eryptosis in patients with CKD stage 3-5 and 5D in our study. Inflammation and elevated parathormone were also associated with increased eryptosis. Therefore, eryptosis may be an important factor in the pathogenesis of renal anaemia. Inflammation associated CKD may increase eryptosis. In addition, this effect can be taken into consideration in the planning of treatment because of the lower incidence of eryptosis in patients using calcium channel blocker and EPO.

Neutral lipids regulate amphipathic helix affinity for model lipid droplets

Cellular lipid droplets (LDs) have a neutral lipid core shielded from the aqueous environment by a phospholipid monolayer containing proteins. These proteins define the biological functions of LDs, and most of them bear amphipathic helices (AH), which can selectively target to LDs, or to LD subsets. How such binding preference happens remains poorly understood. Here, we found that artificial LDs made of different neutral lipids but presenting equal phospholipid packing densities differentially recruit AHs. Varying the phospholipid density shifts the binding levels, but the differential recruitment is unchanged. We found that the binding level of AHs is defined by their interaction preference with neutral lipids and ability to decrease surface tension. The phospholipid packing level regulates mainly the amount of neutral lipid accessible. Therefore, it is the hydrophobic nature of the phospholipid packing voids that controls the binding level of AHs. Our data bring us a major step closer to understanding the binding selectivity of AHs to lipid membranes.

Probing the specificity of CYP112 in bacterial gibberellin biosynthesis

Biosynthesis of the gibberellin A (GA) plant hormones evolved independently in plant-associated fungi and bacteria. While the relevant enzymes have distinct evolutionary origins, the pathways proceed via highly similar reactions. One particularly complex transformation involves combined demethylation and γ-lactone ring formation, catalyzed in bacteria by the cytochrome P450 CYP112 in three individual steps, which involves large structural changes in the transition from substrate to product, with further divergence in the recently demonstrated use of two separate mechanistic routes. Here, the substrate specificity of the isozyme from Erwinia tracheiphila, EtCYP112, was probed via UV–Vis spectral binding studies and activity assays with alternate substrates from the GA biosynthetic pathway. EtCYP112 tightly binds its native substrate GA12 and reaction intermediates GA15 and GA24, as well as the methylated derivatives of GA12 and GA15. It, however, only poorly binds methylated GA24, its GA9 final product and the C-20 carboxylate side product GA25. These distinct affinities are consistent with the known reactivity of EtCYP112. However, while it binds to the immediately preceding pathway metabolite GA12-aldehyde and even earlier oxygenated ent-kaurene precursors, EtCYP112 only reacts with GA12-aldehyde and not the earlier ent-kaurene-derived metabolites. Even with GA12-aldehyde conversion is limited to the first two steps, and the full combined demethylation and γ-lactone ring-forming transformation is not catalyzed. Thus, CYP112 has evolved specificity at the catalytic rather than substrate-binding level to enable its role in GA biosynthesis.

Decreased transcription factor binding levels nearby primate pseudogenes suggests regulatory degeneration

Characteristics of pseudogene degeneration at the coding level are well-known, such as a shift towards neutral rates of nonsynonymous substitutions and gain of frameshift mutations. In contrast, degeneration of pseudogene transcriptional regulation is not well understood. Here, we test two predictions of regulatory degeneration along the pseudogenized lineage: (1) decreased transcription factor binding and (2) accelerated evolution in putative cis-regulatory regions. We find evidence for decreased TF binding levels nearby two primate pseudogenes compared to functional liver genes. We also find evidence for pseudogene-lineage-specific relaxation of sequence constraint on a fragment of the promoter of the primate pseudogene urate oxidase (Uox) and a nearby cis-regulatory module (CRM). However, the majority of TF-bound sequences nearby pseudogenes do not show evidence for lineage-specific accelerated rates of evolution. We conclude that decreases in TF binding level could be a marker for regulatory degeneration, while sequence degeneration in most CRMs may be obscured by background rates of TF binding site turnover.

Platelet Desialylation Is Closely Associated with Cytotoxic T Lymphocyte-Mediated Platelet Destruction in Immune Thrombocytopenia

Introduction: ITP is an autoimmune hemorrhagic disorder characterized by increased platelet destruction and insufficient platelet production. The classical mechanisms of platelet destruction include premature platelet clearance mediated by antiplatelet autoantibodies in the reticuloendothelial system and cytotoxic T lymphocyte (CTL)-mediated platelet lysis. Platelet desialylation, the process of removing sialic acid residues from glycoproteins, which results in the exposure of beta-galactose on platelet surface, can initiate platelet recognition by asialoglycoprotein receptors (ASGPRs)-expressing liver macrophages and hepatocytes and then lead to platelet clearance in the liver. The status and role of platelet desialylation in ITP have not been studied yet. In this study, we detected the level of platelet desialylation and analyzed its association with antiplatelet autoantibody- and CTL- mediated platelet destruction in ITP. Methods: Ethylenediaminetetraacetic acid anti-coagulated whole blood was obtained from 74 active ITP patients and 34 matched controls. Platelets, CD8+ T lymphocytes and plasma were isolated. The levels of platelet desialylation in ITP and controls were evaluated by the binding level of FITC conjugated Ricinus communis agglutinin I (RCA-I) on platelets by flow cytometry. In addition, the modified antibody-specific immobilization of platelet antigens (MAIPA) and assay of CTL-mediated cytotoxicity towards platelets were performed to evaluate antiplatelet autoantibody- and CTL-mediated platelet destruction in ITP patients. Results: Our results showed that the binding level of RCA-I on platelets was significantly elevated in ITP patients compared with controls (Figure 1). Among these 74 ITP patients, 10 were single anti-GPIIb/IIIa positive, 13 were single anti-GPIb positive, 16 were positive for both, and 35 were negative for autoantibodies. There was neither significant difference in the binding level of RCA-I on platelets between patients with anti-GPIIb/IIIa and those without anti-GPIIb/IIIa antibodies, nor between patients with anti-GPIb and those without anti-GPIb antibodies (data not shown). Moreover, we conducted the assay of CTL-mediated cytotoxicity towards platelets in 40 ITP patients and 13 controls. Positively induced platelet apoptosis (greater than mean plus 2 SD for the controls) was detected in 22 of 40 ITP patients, which was regarded as group A. The other 18 patients was referred to as group B. Interestingly, the binding level of RCA-I was significantly higher in group A than that in group B (Figure 2). These findings indicated that the level of platelet desialylation was markedly elevated in ITP patients than controls, and significantly higher in ITP patients with CTL-mediated platelet destruction. We supposed that neuraminidase-1, which could translocate to platelet surface after platelet lesion and cause platelet desialylation, was elevated from an internal platelet store after CTL-mediated platelet destruction, and then caused increased beta-galactose exposure and led to further platelet clearance in the liver. The specific mechanism still needs further study. Conclusion: In conclusion, our results suggest that platelet desialylation may be involved in the pathogenesis of ITP and closely associated with CTL-mediated platelet destruction. Inhibiting platelet desialylation may be a novel therapeutic strategy for ITP patients with CTL-mediated platelet destruction. Figure 1 The level of platelet desialylation in ITP patients and controls. A. The representative flow cytometry results. B. The binding level of RCA-I was higher in ITP patients than controls (***P < 0.001). Figure 1. The level of platelet desialylation in ITP patients and controls. A. The representative flow cytometry results. B. The binding level of RCA-I was higher in ITP patients than controls (***P < 0.001). Figure 2 Figure 2. The level of platelet desialylation was higher in ITP patients with CTL-mediated platelet destruction. A. Induced platelet apoptosis by CTLs in controls, group A and group B. B. The representative scattergrams of platelet apoptosis. Dots in the lower right corner represented apoptotic platelets. C. The binding level of RCA-I was significantly higher in group A or B than controls (***P < 0.001; *P = 0.042). Moreover, it was higher in group A than group B (*P = 0.015). Disclosures No relevant conflicts of interest to declare.

A Dataset of Experimental HLA-B*2705 Peptide Binding Affinities

T-cell epitopes form the basis of many vaccines, diagnostics, and reagents. Current methods for the in silico identification of T-cell epitopes rely, in the main, on the accurate quantitative prediction of peptide-Major Histocompatibility Complex (pMHC) affinity using data-driven computational approaches. Here, we describe a dataset of experimentally determined pMHC binding affinities for the problematic human class I allele HLA-B*2705. Using an in-house, FACS-based, MHC stabilization assay, we measured binding of 223 peptides. This dataset includes both nonbinding and binding peptides, with measured affinities (expressed as −log10 of the half-maximal binding level) ranging from 1.2 to 7.4. This dataset should provide a useful independent benchmark for new and existing methods for predicting peptide binding to HLA-B*2705.