Role of Dnl4-Lif1 in nonhomologous end-joining repair complex assembly and suppression of homologous recombination

Nonhomologous end joining (NHEJ) eliminates DNA double-strand breaks (DSBs) in bacteria and eukaryotes. In Saccharomyces cerevisiae, there are pairwise physical interactions among the core complexes of the NHEJ pathway, namely Yku70-Yku80 (Ku), Dnl4-Lif1 and Mre11-Rad50-Xrs2 (MRX). However, MRX also has a key role in the repair of DSBs by homologous recombination (HR). Here we have examined the assembly of NHEJ complexes at DSBs biochemically and by chromatin immunoprecipitation. Ku first binds to the DNA end and then recruits Dnl4-Lif1. Notably, Dnl4-Lif1 stabilizes the binding of Ku to in vivo DSBs. Ku and Dnl4-Lif1 not only initiate formation of the nucleoprotein NHEJ complex but also attenuate HR by inhibiting DNA end resection. Therefore, Dnl4-Lif1 plays an important part in determining repair pathway choice by participating at an early stage of DSB engagement in addition to providing the DNA ligase activity that completes NHEJ.     

PNUTS,a protein phosphatase 1 (PP1) nuclear targeting subunit. Characterization of its PP1- and RNA-binding domains and regulation by phosphorylation

PNUTS, Phosphatase 1 NUclear Targeting Subunit, is a recently described protein that targets protein phosphatase 1 (PP1) to the nucleus. In the present study, we characterized the biochemical properties of PNUTS. A variety of truncation and site-directed mutants of PNUTS was prepared and expressed either as glutathione S-transferase fusion proteins in Escherichia coli or as FLAG-tagged proteins in 293T cells. A 50-amino acid domain in the center of PNUTS mediated both high affinity PP1 binding and inhibition of PP1 activity. The PP1-binding domain is related to a motif found in several other PP1-binding proteins but is distinct in that Trp replaces Phe. Mutation of the Trp residue essentially abolished the ability of PNUTS to bind to and inhibit PP1. The central PP1-binding domain of PNUTS was an effective substrate for protein kinase A in vitro, and phosphorylation substantially reduced the ability of PNUTS to bind to PP1 in vitro and following stimulation of protein kinase A in intact cells. In vitro RNA binding experiments showed that a C-terminal region including several RGG motifs and a novel repeat domain rich in His and Gly interacted with mRNA and single-stranded DNA. PNUTS exhibited selective binding for poly(A) and poly(G) compared with poly(U) or poly(C) ribonucleotide homopolymers, with specificity being mediated by distinct regions within the domain rich in His and Gly and the domain containing the RGG motifs. Finally, a PNUTS-PP1 complex was isolated from mammalian cell lysates using RNA-conjugated beads. Together, these studies support a role for PNUTS in protein kinase A-regulated targeting of PP1 to specific RNA-associated complexes in the nucleus.     

Model for membrane organization and protein sorting in the cyanobacterium Synechocystis sp. PCC 6803 inferred from proteomics and multivariate sequence analyses

Cyanobacteria are unique eubacteria with an organized subcellular compartmentalization of highly differentiated internal thylakoid membranes (TM), in addition to the outer and plasma membranes (PM). This leads to a complicated system for transport and sorting of proteins into the different membranes and compartments. By shotgun and gel-based proteomics of plasma and thylakoid membranes from the cyanobacterium Synechocystis sp. PCC 6803, a large number of membrane proteins were identified. Proteins localized uniquely in each membrane were used as a platform describing a model for cellular membrane organization and protein intermembrane sorting and were analyzed by multivariate sequence analyses to trace potential differences in sequence properties important for insertion and sorting to the correct membrane. Sequence traits in the C-terminal region, but not in the N-terminal nor in any individual transmembrane segments, were discriminatory between the TM and PM classes. The results are consistent with a contact zone between plasma and thylakoid membranes, which may contain short-lived "hemifusion" protein traffic connection assemblies. Insertion of both integral and peripheral membrane proteins is suggested to occur through common translocons in these subdomains, followed by a potential translation arrest and structure-based sorting into the correct membrane compartment.     

Growth-promoting effects of lactoferrin on L. acidophilus and Bifidobacterium spp.

We investigated the effects of lactoferrin on the growth of L. acidophilus CH-2, Bifidobacterium breve ATCC 15700, B. longum ATCC 15707, B. infantis ATCC 15697, and B. bifidum ATCC 15696. The growth of L. acidophilus was stimulated by bovine holo-lactoferrin but not by apo-lactoferrin. With bifidobacteria, bovine lactoferrin stimulated growth of three strains: B. breve, B. infantis and B. bifidum under certain conditions. Both apoprotein and holoprotein had similar effects. However, B. longum growth was not affected by lactoferrin. Thus, the mechanism of stimulating growth of bifidobacteria may be different from that of L. acidophilus. By far-western blotting using biotinylated lactoferrin and horseradish peroxidase-conjugated streptavidin, lactoferrin-binding proteins were detected in the membrane protein fraction of L. acidophilus, B. bifidum, B. infantis and B. breve. The molecular weights of lactoferrin-binding proteins of L. acidophilus were estimated from SDS-polyacrylamide gel electrophoresis to be 27, 41 and 67 kDa, and those of the three bifidobacterial strains were estimated to be 67-69 kDa. However, no such lactoferrin-binding components were detected in the membrane fraction of B. longum. It is interesting that the appearance of lactoferrin-binding proteins in the membrane fraction of these species corresponds to their growth stimulation by lactoferrin.     

Cortical tissue-specific accumulation of the root-specific ns-LTP transcripts in the bean (Phaseolus vulgaris) seedlings

The characterization of a cDNA clone encoding non-specific lipid transfer protein (PvLTP, formerly named PVR3) in the roots of bean seedlings has been previously reported. In this study, we examined the temporal and spatial accumulation of PvLTP mRNA and the effect of the auxin naphthaleneacetic acid (NAA) on the accumulation of PvLTP mRNA during root development. In situ hybridization showed that accumulation of PvLTP mRNA is highly tissue-specific. Accumulation was detected in the cortical tissue, but not in other tissues of root, including the quiescent center and root cap. Within the cortical tissue, accumulation of PvLTP mRNA was developmentally regulated; accumulation of PvLTP mRNA was high in the cortical tissue of the proximal and ground meristem and declined as cortical tissue developed further. Since the appropriate distribution of auxin is an important factor responsible for the maintenance of root meristem organization. We examined effect of auxin on the accumulation of PvLTP mRNA in relation to the development of cortical tissue. In bean seedlings grown on medium supplemented with 5 M NAA, morphological alternations, including radial root expansion and abnormal tissue organization in the root apical meristem, were observed. Only faint accumulation signals of PvLTP mRNA were observed in the cortical tissue of proximal meristem region, indicating that cortical tissue development was repressed by exogenous NAA. However, our results suggest that the change in accumulation of PvLTP mRNA is not direct regulatory effect but reflective effect of altered development of cortical tissue that was induced by exogenous NAA. The temporal and spatial accumulation of PvLTP mRNA indicates that PvLTP is a useful marker for the development of cortical tissue in the root tip in bean seedlings.     

AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

The phosphoinositide 3-kinase–Akt signaling pathway is essential to many biological processes, including cell proliferation, survival, metabolism, and angiogenesis, under pathophysiological conditions. Although 3-phosphoinositide–dependent kinase 1 (PDK1) is a primary activator of Akt at the plasma membrane, the optimal activation mechanism remains unclear. We report that adhesion molecule with IgG-like domain 2 (AMIGO2) is a novel scaffold protein that regulates PDK1 membrane localization and Akt activation. Loss of AMIGO2 in endothelial cells (ECs) led to apoptosis and inhibition of angiogenesis with Akt inactivation. Amino acid residues 465–474 in AMIGO2 directly bind to the PDK1 pleckstrin homology domain. A synthetic peptide containing the AMIGO2 465–474 residues abrogated the AMIGO2–PDK1 interaction and Akt activation. Moreover, it effectively suppressed pathological angiogenesis in murine tumor and oxygen-induced retinopathy models. These results demonstrate that AMIGO2 is an important regulator of the PDK1–Akt pathway in ECs and suggest that interference of the PDK1–AMIGO2 interaction might be a novel pharmaceutical target for designing an Akt pathway inhibitor.     

Effectiveness of autologous serum as an alternative to fetal bovine serum in adipose-derived stem cell engineering

In cell culture, medium supplemented with fetal bovine serum is commonly used, and it is widely known that fetal bovine serum supplies an adequate environment for culture and differentiation of stem cells. Nevertheless, the use of xenogeneic serum can cause several problems. We compared the effects of four different concentrations of autologous serum (1, 2, 5, and 10 %) on expansion and adipogenic differentiation of adipose-derived stem cells using 10 % fetal bovine serum as a control. The stem cells were grafted on nude mice and the in vivo differentiation capacity was evaluated. The isolation of adipose-derived stem cells was successful irrespective of the culture medium. The proliferation potential was statistically significant at passage 2, as follows: 10 % autologous serum >10 % fetal bovine serum = 5 % autologous serum >2 % autologous serum = 1 % autologous serum. The differentiation capacity appeared statistically significant at passage 4, as follows: 10 % fetal bovine serum >10 % autologous serum = 5 % autologous serum >2 % autologous serum = 1 % autologous serum. Ten percent autologous serum and 10 % fetal bovine serum had greater differentiation capacity than 1 and 2 % autologous serum in vivo, and no significant difference was observed between the groups at ≥5 % concentration at 14 weeks. In conclusion, 10 % autologous serum was at least as effective as 10 % fetal bovine serum with respect to the number of adipose-derived stem cells at the end of both isolation and expansion, whereas 1 and 2 % autologous serum was inferior.     

Differential desensitization and internalization of three different bullfrog gonadotropin-releasing hormone receptors

We previously demonstrated the presence of three distinct types of the gonadotropin-releasing hormone receptor (GnRHR) in a bullfrog (denoted bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3). The bfGnRHRs exhibited differential tissue distribution and ligand selectivity. In the present study, we demonstrated the desensitization and internalization kinetics of these receptors in both transiently-transfected HEK293 cells and retrovirus-mediated stable cells. The time-course accumulation of the inositol phosphate in response to GnRH revealed that bfGnRHR-1 and -2 were rapidly desensitized, whereas bfGnRHR-3 was slowly desensitized. A comparison of the internalization kinetics revealed the most rapid rate and highest extent of internalization of bfGnRHR-2 among the three receptors. Interestingly, the mechanisms that underlie the receptor internalization appear to differ from each other. Internalization of bfGnRHR-1 was dependent on both dynamin and beta-arrestin, whereas those of bfGnRHR-2 and -3 were dependent on dynamin, but not on arrestin. These results, therefore, suggest that differential regulatory mechanisms for desensitization and internalization of the GnRHR are involved in diverse cellular and physiological responses to GnRH stimulation.     

Spatial patterns,ecological niches,and interspecific competition of avian brood parasites: inferring from a case study of Korea

Since obligate avian brood parasites depend completely on the effort of other host species for rearing their progeny, the availability of hosts will be a critical resource for their life history. Circumstantial evidence suggests that intense competition for host species may exist not only within but also between species. So far, however, few studies have demonstrated whether the interspecific competition really occurs in the system of avian brood parasitism and how the nature of brood parasitism is related to their niche evolution. Using the occurrence data of five avian brood parasites from two sources of nationwide bird surveys in South Korea and publically available environmental/climatic data, we identified their distribution patterns and ecological niches, and applied species distribution modeling to infer the effect of interspecific competition on their spatial distribution. We found that the distribution patterns of five avian brood parasites could be characterized by altitude and climatic conditions, but overall their spatial ranges and ecological niches extensively overlapped with each other. We also found that the predicted distribution areas of each species were generally comparable to the realized distribution areas, and the numbers of individuals in areas where multiple species were predicted to coexist showed positive relationships among species. In conclusion, despite following different coevolutionary trajectories to adapt to their respect host species, five species of avian brood parasites breeding in South Korea occupied broadly similar ecological niches, implying that they tend to conserve ancestral preferences for ecological conditions. Furthermore, our results indicated that contrary to expectation interspecific competition for host availability between avian brood parasites seemed to be trivial, and thus, play little role in shaping their spatial distributions and ecological niches. Future studies, including the complete ranges of avian brood parasites and ecological niches of host species, will be worthwhile to further elucidate these issues.