MDC1 Interacts with TOPBP1 to Maintain Chromosomal Stability during Mitosis.

In mitosis, cells inactivate DNA double-strand break (DSB) repair pathways to preserve genome stability. However, some early signaling events still occur, such as recruitment of the scaffold protein MDC1 to phosphorylated histone H2AX at DSBs. Yet, it remains unclear whether these events are important for maintaining genome stability during mitosis. Here, we identify a highly conserved protein-interaction surface in MDC1 that is phosphorylated by CK2 and recognized by the DNA-damage response mediator protein TOPBP1. Disruption of MDC1-TOPBP1 binding causes a specific loss of TOPBP1 recruitment to DSBs in mitotic but not interphase cells, accompanied by mitotic radiosensitivity, increased micronuclei, and chromosomal instability. Mechanistically, we find that TOPBP1 forms filamentous structures capable of bridging MDC1 foci in mitosis, indicating that MDC1-TOPBP1 complexes tether DSBs until repair is reactivated in the following G1 phase. Thus, we reveal an important, hitherto-unnoticed cooperation between MDC1 and TOPBP1 in maintaining genome stability during cell division.

NBS1 promotes the endonuclease activity of the MRE11-RAD50 complex by sensing CtIP phosphorylation.

DNA end resection initiates DNA double-strand break repair by homologous recombination. MRE11-RAD50-NBS1 and phosphorylated CtIP perform the first resection step via MRE11-catalyzed endonucleolytic DNA cleavage. Human NBS1, more than its homologue Xrs2 in Saccharomyces cerevisiae, is crucial for this process, highlighting complex mechanisms that regulate the MRE11 nuclease in higher eukaryotes. Using a reconstituted system, we show here that NBS1, through its FHA and BRCT domains, functions as a sensor of CtIP phosphorylation. NBS1 then activates the MRE11-RAD50 nuclease through direct physical interactions with MRE11. In the absence of NBS1, MRE11-RAD50 exhibits a weaker nuclease activity, which requires CtIP but not strictly its phosphorylation. This identifies at least two mechanisms by which CtIP augments MRE11: a phosphorylation-dependent mode through NBS1 and a phosphorylation-independent mode without NBS1. In support, we show that limited DNA end resection occurs in vivo in the absence of the FHA and BRCT domains of NBS1. Collectively, our data suggest that NBS1 restricts the MRE11-RAD50 nuclease to S-G2 phase when CtIP is extensively phosphorylated. This defines mechanisms that regulate the MRE11 nuclease in DNA metabolism.

Heparanase expression in term placentas of diabetic patients and healthy controls.

PURPOSE: The prevalence of diabetic disorders in pregnancy is rising, which goes along with increased risks for maternal and foetal complications during pregnancy and delivery. The expression of the endo-ß-D: -glucuronidase, heparanase (HPSE), may increase under hyperglycaemic conditions, is believed to play an important role in diabetes associated morbidity outside the female reproductive tract and is expressed in the placenta throughout gestation. However, the placental expression of HPSE has not been investigated in diabetic patients. MATERIALS AND METHODS: Placental biopsies of 30 patients with pre-existing or gestational diabetes and 30 healthy controls were analysed by real-time PCR and immunohistochemistry with regard to the presence of HPSE at term. RESULTS: Patients and controls were comparable with respect to foetal outcome and maternal characteristics except for maternal body mass index. We were unable to show significant differences in placental HPSE expression between diabetic patients and healthy controls. DISCUSSION: This study suggests that HPSE expression in term placentas is not affected by maternal diabetes and thus does not contribute to pathological processes in diabetic pregnancies with deliveries at term.

Growth-modulatory effects of heparin and VEGF165 on the choriocarcinoma cell-line JEG-3 and its expression of heparanase.

BACKGROUND: Expression of heparanase (HPSE) in tumor cells is strongly associated with invasion, metastasis and angiogenesis. It also plays a key role during pregnancy, in processes of implantation as well as placentation. Vascular endothelial growth factor (VEGF) and heparin are known to alter HPSE expression, with heparin given prophylactically to women with a history of placenta-mediated complications in subsequent pregnancies. MATERIALS AND METHODS: We examined the growth-modulatory effects of different concentrations of heparin and VEGF on the choriocarcinoma cell-line JEG-3 and the expression of heparanase under VEGF and heparin by proliferation assays, PCR, and western blot. RESULTS: Proliferation of JEG-3 cells was induced by heparin in a dose-dependent manner, whereas highly concentrated VEGF led to a decreased cell proliferation. Both agents did not influence the HPSE-expression. CONCLUSION: The presumed pregnancy-protecting effects of heparin may partially be due to an increase of trophoblast proliferation and not via regulation of HPSE expression.

Turnover of amyloid precursor protein family members determines their nuclear signaling capability.

The amyloid precursor protein (APP) as well as its homologues, APP-like protein 1 and 2 (APLP1 and APLP2), are cleaved by α-, ß-, and γ-secretases, resulting in the release of their intracellular domains (ICDs). We have shown that the APP intracellular domain (AICD) is transported to the nucleus by Fe65 where they jointly bind the histone acetyltransferase Tip60 and localize to spherical nuclear complexes (AFT complexes), which are thought to be sites of transcription. We have now analyzed the subcellular localization and turnover of the APP family members. Similarly to AICD, the ICD of APLP2 localizes to spherical nuclear complexes together with Fe65 and Tip60. In contrast, the ICD of APLP1, despite binding to Fe65, does not translocate to the nucleus. In addition, APLP1 predominantly localizes to the plasma membrane, whereas APP and APLP2 are detected in vesicular structures. APLP1 also demonstrates a much slower turnover of the full-length protein compared to APP and APLP2. We further show that the ICDs of all APP family members are degraded by the proteasome and that the N-terminal amino acids of ICDs determine ICD degradation rate. Together, our results suggest that different nuclear signaling capabilities of APP family members are due to different rates of full-length protein processing and ICD proteasomal degradation. Our results provide evidence in support of a common nuclear signaling function for APP and APLP2 that is absent in APLP1, but suggest that APLP1 has a regulatory role in the nuclear translocation of APP family ICDs due to the sequestration of Fe65.