Functional regulation of FEN1 nuclease and its link to cancer

Flap endonuclease-1 (FEN1) is a member of the Rad2 structure-specific nuclease family. FEN1 possesses FEN, 5'-exonuclease and gap-endonuclease activities. The multiple nuclease activities of FEN1 allow it to participate in numerous DNA metabolic pathways, including Okazaki fragment maturation, stalled replication fork rescue, telomere maintenance, long-patch base excision repair and apoptotic DNA fragmentation. Here, we summarize the distinct roles of the different nuclease activities of FEN1 in these pathways. Recent biochemical and genetic studies indicate that FEN1 interacts with more than 30 proteins and undergoes post-translational modifications. We discuss how FEN1 is regulated via these mechanisms. Moreover, FEN1 interacts with five distinct groups of DNA metabolic proteins, allowing the nuclease to be recruited to a specific DNA metabolic complex, such as the DNA replication machinery for RNA primer removal or the DNA degradosome for apoptotic DNA fragmentation. Some FEN1 interaction partners also stimulate FEN1 nuclease activities to further ensure efficient action in processing of different DNA structures. Post-translational modifications, on the other hand, may be critical to regulate protein-protein interactions and cellular localizations of FEN1. Lastly, we also review the biological significance of FEN1 as a tumor suppressor, with an emphasis on studies of human mutations and mouse models.     

Synthesis and biological evaluation of glycogen synthase kinase 3 (GSK-3) inhibitors: an fast and atom efficient access to 1-aryl-3-benzylureas

The glycogen synthase kinase 3 (GSK-3) is implicated in multiple cellular processes and has been linked to the pathogenesis of Alzheimer's disease (AD). In the course of our research topic we synthesized a library of potent GSK-3 inhibitors. We utilized the urea scaffold present in the potent and highly selective GSK-3 inhibitor AR-A014418 (AstraZeneca). This moiety suits both (a) a convergent approach utilizing readily accessible building blocks and (b) a divergent approach based on a microwave heating assisted Suzuki coupling. We established a chromatography-free purification method to generate products with sufficient purity for the biological assays. The structure-activity relationship of the library provided the rationale for the synthesis of the benzothiazolylurea 66 (IC(50)=140 nM) and the pyridylurea 62 (IC(50)=98 nM), which displayed two to threefold enhanced activity versus the reference compound 18 (AR-A014418: IC(50)=330 nM) in our assays.     

Distinct molecular regulation of glycogen synthase kinase-3alpha isozyme controlled by its N-terminal region: functional role in calcium/calpain signaling

Glycogen synthase kinase-3 (GSK-3) is expressed as two isozymes α and β. They share high similarity in their catalytic domains but differ in their N- and C-terminal regions, with GSK-3α having an extended glycine-rich N terminus. Here, we undertook live cell imaging combined with molecular and bioinformatic studies to understand the distinct functions of the GSK-3 isozymes focusing on GSK-3α N-terminal region. We found that unlike GSK-3β, which shuttles between the nucleus and cytoplasm, GSK-3α was excluded from the nucleus. Deletion of the N-terminal region of GSK-3α resulted in nuclear localization, and treatment with leptomycin B resulted in GSK-3α accumulation in the nucleus. GSK-3α rapidly accumulated in the nucleus in response to calcium or serum deprivation, and accumulation was strongly inhibited by the calpain inhibitor calpeptin. This nuclear accumulation was not mediated by cleavage of the N-terminal region or phosphorylation of GSK-3α. Rather, we show that calcium-induced GSK-3α nuclear accumulation was governed by GSK-3α binding with as yet unknown calpain-sensitive protein or proteins; this binding was mediated by the N-terminal region. Bioinformatic and experimental analyses indicated that nuclear exclusion of GSK-3α was likely an exclusive characteristic of mammalian GSK-3α. Finally, we show that nuclear localization of GSK-3α reduced the nuclear pool of β-catenin and its target cyclin D1. Taken together, these data suggest that the N-terminal region of GSK-3α is responsible for its nuclear exclusion and that binding with a calcium/calpain-sensitive product enables GSK-3α nuclear retention. We further uncovered a novel link between calcium and nuclear GSK-3α-mediated inhibition of the canonical Wnt/β-catenin pathway.     

Selective loss of glycogen synthase kinase-3α in birds reveals distinct roles for GSK-3 isozymes in tau phosphorylation

Mammalian glycogen synthase kinase-3 (GSK-3), a critical regulator in neuronal signaling, cognition, and behavior, exists as two isozymes GSK-3α and GSK-3β. Their distinct biological functions remains largely unknown. Here, we examined the evolutionary significance of each of these isozymes. Surprisingly, we found that unlike other vertebrates that harbor both GSK-3 genes, the GSK-3α gene is missing in birds. GSK-3-mediated tau phosphorylation was significantly lower in adult bird brains than in mouse brains, a phenomenon that was reproduced in GSK-3α knockout mouse brains. Tau phosphorylation was detected in brains from bird embryos suggesting that GSK-3 isozymes play distinct roles in tau phosphorylation during development. Birds are natural GSK-3α knockout organisms and may serve as a novel model to study the distinct functions of GSK-3 isozymes.     

Elucidating substrate and inhibitor binding sites on the surface of GSK-3β and the refinement of a competitive inhibitor

A molecular understanding of substrate recognition of protein kinases provides an important basis for the development of substrate competitive inhibitors. Here, we explored substrate recognition and competitive inhibition of glycogen synthase kinase (GSK)-3β using molecular and computational tools. In previous work, we described Gln89 and Asn95 within GSK-3β as important substrates binding sites. Here, we show that the cavity bordered by loop 89-QDKRFKN-95, located in the vicinity of the GSK-3β catalytic core, is a promiscuous substrate binding subsite. Mutations within this segment highlighted Phe93 as an additional essential contact residue for substrates' recognition. However, unlike Gln89 and Asn95, Phe93 was also important for the binding of our previously described substrate competitive inhibitor, L803 [KEAPPAPPQS(p)P], and its cell-permeable variant L803-mts. The effects of the substitution of charged or polar residues within L803 further suggested that binding to GSK-3β is governed by hydrophobic interactions. Our computational model of GSK-3β bound to L803 was in agreement with the experimental data. It revealed L803 binding with a hydrophobic surface patch and identified interactions between Pro8 (L803) and Phe93 (GSK-3β). Computational modeling of new L803 variants predicted that inhibition would be strengthened by adding contacts with Phe93 or by increasing the hydrophobic content of the peptide. Indeed, the newly designed L803 variants showed improved inhibition. Our study identified different and overlapping elements in GSK-3β substrate and inhibitor recognition and provides a novel example for model-based rational design of substrate competitive inhibitors for GSK-3.     

Adjuvant Docetaxel and Cyclophosphamide (DC) with Prophylactic Granulocyte Colony-Stimulating Factor (G-CSF) on Days 8 &12 in Breast Cancer Patients: A Retrospective Analysis

Purpose

Four cycles of docetaxel/cyclophosphamide (DC) resulted in superior survival than doxorubicin/cyclophosphamide in the treatment of early breast cancer. The original study reported a 5% incidence of febrile neutropenia (FN) recommending prophylactic antibiotics with no granulocyte colony-stimulating factor (G-CSF) support. The worldwide adoption of this protocol yielded several reports on substantially higher rates of FN events. We explored the use of growth factor (GF) support on days 8 and 12 of the cycle with the original DC protocol.

Methods

Our study included all consecutive patients with stages I–II breast cancer who were treated with the DC protocol at the Institute of Oncology, Davidoff Center (Rabin Medical Center, Petah Tikva, Israel) from April, 2007 to March, 2012. Patient, tumor characteristics, and toxicity were reported. Results: In total, 123 patients received the DC regimen. Median age was 60 years, (range, 25–81 years). Thirty-three patients (26.8%) were aged 65 years and older. Most of the women (87%) adhered to the planned G-CSF protocol (days 8 &12). 96% of the patients completed the 4 planned cycles of chemotherapy. Six patients (5%) had dose reductions, 6 (5%) had treatment delays due to non-medical reasons. Thirteen patients (10.6%) experienced at least one event of FN (3 patients had 2 events), all requiring hospitalization. Eight patients (6.5%) required additional support with G-CSF after the first chemotherapy cycle, 7 because of FN and one due to neutropenia and diarrhea.

In Conclusion

Primary prophylactic G-CSF support on days 8 and 12 of the cycle provides a tolerable option to deliver the DC protocol. Our results are in line with other retrospective protocols using longer schedules of GF support.     

Reproductive and metabolic responses of desert adapted common spiny male mice (Acomys cahirinus) to vasopressin treatment

Sufficient amounts of water and food are important cues for reproduction in an unpredictable environment. We previously demonstrated that increased osmolarity levels, or exogenous vasopressin (VP) treatment halt reproduction of desert adapted golden spiny mice Acomys russatus. In this research we studied gonad regulation by VP and food restriction (FR) in desert adapted common spiny mouse (A. cahirinus) males, kept under two different photoperiod regimes-short (SD-8L:16D) and long (LD-16L:8D) days. Mice were treated with VP, FR, and VP+FR for three weeks. Response was assessed from changes in relative testis mass, serum testosterone levels and mRNA receptor gene expression of VP, aldosterone and leptin in treated groups, compared with their controls. SD-acclimation increased testosterone levels, VP treatment decreased expression of aldosterone mRNA receptor in the testes of SD-acclimated males. FR under SD-conditions resulted in testosterone decrease and elevation of VP- receptor gene expression in testes. Aldosterone receptor mRNA expression was also detected in WAT. These results support the idea that water and food availability in the habitat may be used as signals for activating the reproductive system through direct effects of VP, aldosterone and leptin on the testes or through WAT by indirect effects.     

Phylodynamics and Dispersal of HRSV Entails Its Permanence in the General Population in between Yearly Outbreaks in Children

Background

Human respiratory syncytial virus (HRSV) is one of the major etiologic agents of respiratory tract infections among children worldwide.

Methodology/Principal Findings

Here through a comprehensive analysis of the two major HRSV groups A and B (n = 1983) which comprise of several genotypes, we present a complex pattern of population dynamics of HRSV over a time period of 50 years (1956–2006). Circulation pattern of HRSV revealed a series of expansions and fluctuations of co-circulating lineages with a predominance of HRSVA. Positively selected amino acid substitutions of the G glycoprotein occurred upon population growth of GB3 with a 60-nucleotide insertion (GB3 Insert), while other genotypes acquired substitutions upon both population growth and decrease, thus possibly reflecting a role for immune selected epitopes in linkage to the traced substitution sites that may have important relevance for vaccine design. Analysis evidenced the co-circulation and predominance of distinct HRSV genotypes in Brazil and suggested a year-round presence of the virus. In Brazil, GA2 and GA5 were the main culprits of HRSV outbreaks until recently, when the GB3 Insert became highly prevalent. Using Bayesian methods, we determined the dispersal patterns of genotypes through several inferred migratory routes.

Conclusions/Significance

Genotypes spread across continents and between neighboring areas. Crucially, genotypes also remained at any given region for extended periods, independent of seasonal outbreaks possibly maintained by re-infecting the general population.     

The ATM-BID pathway regulates quiescence and survival of haematopoietic stem cells

BID, a BH3-only BCL2 family member, functions in apoptosis as well as the DNA-damage response. Our previous data demonstrated that BID is an ATM effector acting to induce cell-cycle arrest and inhibition of apoptosis following DNA damage. Here we show that ATM-mediated BID phosphorylation plays an unexpected role in maintaining the quiescence of haematopoietic stem cells (HSCs). Loss of BID phosphorylation leads to escape from quiescence of HSCs, resulting in exhaustion of the HSC pool and a marked reduction of HSC repopulating potential in vivo. We also demonstrate that BID phosphorylation plays a role in protecting HSCs from irradiation, and that regulating both quiescence and survival of HSCs depends on BID's ability to regulate oxidative stress. Moreover, loss of BID phosphorylation, ATM knockout or exposing mice to irradiation leads to an increase in mitochondrial BID, which correlates with an increase in mitochondrial oxidative stress. These results show that the ATM-BID pathway serves as a critical checkpoint for coupling HSC homeostasis and the DNA-damage stress response to enable long-term regenerative capacity.