Effects of single-stranded DNA binding proteins on primer extension by telomerase

We present a biochemical analysis of the effects of three single-stranded DNA binding proteins on extension of oligonucleotide primers by the Tetrahymena telomerase. One of them, a human protein designated translin, which was shown to specifically bind the G-rich Tetrahymena and human telomeric repeats, slightly stimulated the primer extension reactions at molar ratios of translin/primer of <1:2. At higher molar ratios, it inhibited the reactions by up to 80%. The inhibition was caused by binding of translin to the primers, rather than by a direct interaction of this protein with telomerase. A second protein, the general human single-stranded DNA binding protein Replication Protein A (RPA), similarly affected the primer extension by telomerase, even though its mode of binding to DNA differs from that of translin. A third protein, the E. coli single-stranded DNA binding protein (SSB), whose binding to DNA is highly cooperative, caused more substantial stimulation and inhibition at the lower and the higher molar ratios of SSB/primer, respectively. Both telomere-specific and general single-stranded DNA binding proteins are found in living cells in telomeric complexes. Based on our data, we propose that these proteins may exert either stimulatory or inhibitory effects on intracellular telomerases, depending on their local concentrations.     

The human protein translin specifically binds single-stranded microsatellite repeats, d(GT)n, and G-strand telomeric repeats, d(TTAGGG)n: a study of the binding parameters

We have previously identified in human fibroblasts a multisubunit protein (designated PGB) that specifically bound single-stranded G-rich microsatellite DNA sequences. PGB was later found to be identical, or closely related to translin, an octameric protein that bound single-stranded DNA consisting of sequences flanking chromosomal translocations. Here, we report that recombinant translin binds single-stranded microsatellite repeats, d(GT)n, and G-strand telomeric repeats, d(TTAGGG)n, with higher affinities (Kdis approximately = 2 nM and Kdis approximately = 12.5 nM, respectively, in 100 mM NaCl and 25 degrees C) than the affinity with which it binds a prototypical sequence flanking translocation sites (Kdis approximately = 23 nM). Translin also binds d(GT)n and d(TTAGGG)n overhangs linked to double-stranded DNA with equilibrium constants in the nanomolar range. Formation of DNA quadruplexes by the d(TTAGGG)n repeats inhibits their binding to translin. A further study of the binding parameters revealed that the minimal length of d(GT)n and d(TTAGGG)n oligonucleotides that a translin octamer can bind is 11 nucleotides, but that such oligonucleotides containing up to 30 nucleotides can bind only a single translin octamer. However, the oligonucleotides d(GT)27 and d(TTAGGG)9 bind two octamers with negative cooperativity. Translin does not detectably bind single-stranded d(GT)n sequences embedded within double-stranded DNA. Based on our data, we propose that translin might be involved in the control of recombination at d(GT)n.d(AC)n microsatellites and in telomere maintenance.     

Intracellular trafficking of vitamin E in hepatocytes: the role of tocopherol transfer protein

The term vitamin E denotes a family of tocopherols and tocotrienols, plant lipids that are essential for vertebrate fertility and health. The principal form of vitamin E found in humans, RRR-alpha-tocopherol (TOH), is thought to protect cells by virtue of its ability to quench free radicals, and functions as the main lipid-soluble antioxidant. Regulation of vitamin E homeostasis occurs in the liver, where TOH is selectively retained while other forms of vitamin E are degraded. Through the action of tocopherol transfer protein (TTP), TOH is then secreted from the liver into circulating lipoproteins that deliver the vitamin to target tissues. Presently, very little is known regarding the intracellular transport of vitamin E. We utilized biochemical, pharmacological, and microscopic approaches to study this process in cultured hepatocytes. We observe that tocopherol-HDL complexes are efficiently internalized through scavenger receptor class B type I. Once internalized, tocopherol arrives within approximately 30 min at intracellular vesicular organelles, where it co-localizes with TTP, and with a marker of the lysosomal compartment (LAMP1), before being transported to the plasma membrane in a TTP-dependent manner. We further show that intracellular processing of tocopherol involves a functional interaction between TTP and an ABC-type transporter.     

Activation of Akt/PKB, increased phosphorylation of Akt substrates and loss and altered distribution of Akt and PTEN are features of Alzheimer's disease pathology

Studies suggest that activation of phosphoinositide 3-kinase-Akt may protect against neuronal cell death in Alzheimer's disease (AD). Here, however, we provide evidence of increased Akt activation, and hyperphosphorylation of critical Akt substrates in AD brain, which link to AD pathogenesis, suggesting that treatments aiming to activate the pathway in AD need to be considered carefully. A different distribution of Akt and phospho-Akt was detected in AD temporal cortex neurons compared with control neurons, with increased levels of active phosphorylated-Akt in particulate fractions, and significant decreases in Akt levels in AD cytosolic fractions, causing increased activation of Akt (phosphorylated-Akt/total Akt ratio) in AD. In concordance, significant increases in the levels of phosphorylation of total Akt substrates, including: GSK3beta(Ser9), tau(Ser214), mTOR(Ser2448), and decreased levels of the Akt target, p27(kip1), were found in AD temporal cortex compared with controls. A significant loss and altered distribution of the major negative regulator of Akt, PTEN (phosphatase and tensin homologue deleted on chromosome 10), was also detected in AD neurons. Loss of phosphorylated-Akt and PTEN-containing neurons were found in hippocampal CA1 at end stages of AD. Taken together, these results support a potential role for aberrant control of Akt and PTEN signalling in AD.     

The SV40 large T-antigen helicase can unwind four stranded DNA structures linked by G-quartets.

We describe a novel activity of the SV40 large T-ag helicase, the unwinding of four stranded DNA structures linked by stacked G-quartets, namely stacked groups of four guanine bases bound by Hoogsteen hydrogen bonds. The structures unwound by the helicase were of two types: (i) quadruplexes comprising four parallel strands that were generated by annealing oligonucleotides including clustered G residues in a buffer containing Na+ions. Each parallel quadruplex consisted of four oligonucleotide molecules. (ii) Complexes comprising two parallel and two antiparallel strands that were generated by annealing the above oligonucleotides in a buffer containing K+ions. Each antiparallel complex consisted of two folded oligonucleotide molecules. Unwinding of these unusual DNA structures by the T-ag was monitored by gel electrophoresis. The unwinding process required ATP and at least one single stranded 3'-tail extending beyond the four stranded region. These data indicated that the T-ag first binds the 3'-tail and moves in a 3'-->5'direction, using energy provided by ATP hydrolysis; then it unwinds the four stranded DNA into single strands. This helicase activity may affect processes such as recombination and telomere extension, in which four stranded DNA could play a role.     

Interleukin-6 and interleukin-6 receptor secretion by chronic lymphatic leukaemia and normal B lymphocytes: effect of PMA and PWM

Interleukin-6 (IL-6) and soluble interleukin-6 receptor (sIL-6R) were detected in supernatants of cultures of B chronic lymphatic leukaemia (CLL) lymphocytes. Phorbol-12-myristate 13 acetate (PMA) caused a decrease in the levels of IL-6 in 14 out of 16 cultures and an increase in levels of sIL6R in all 15 cases. The effect of pokeweed mitogen (PWM) was variable and not significant. The levels of IL-6 were below the detection limit (60 pg/ml) in sera of 13 CLL patients whereas sIL-6R was detected (13 ng/ml to 97 ng/ml) in the 13 sera. IL6 was not detected in cultures of unstimulated or stimulated with PMA or PWM normal human B cells. Levels of sIL-6R were minimal in cultures of normal B lymphocytes and were increased in PMA stimulated cultures. The results are consistent with the view that B-CLL cells produce spontaneously IL-6 which could act in an autocrine fashion to cause shedding of surface IL-6R and account for the correlation found between serum levels of sIL-6R and B-CLL lymphocyte numbers. The fall in levels of IL-6 in PMA stimulated CLL cultures might express masking or degradation of IL-6 after combination with the receptor.     

Approach to discovering novel therapeutic agents for osteoporosis based on integrin receptor blockade

On a global scale, osteoporosis is a major and growing public health problem. In the United States, osteoporosis is present in 24 million people (mostly women) and contributes to more than 1.3 million fractures/year. Serious morbidity and mortality result from these fractures. Current therapies for osteoporosis are few, efficacy is limited, and side effects problematic. Fundamental to the pathophysiology of osteoporosis is an imbalance between the tightly coupled processes of bone resorption and bone formation that characterize normal bone remodeling. Our laboratory is engaged in a research effort focused on elucidating the role of the osteoclast integrin in bone resorption, defining the nature of ligand–integrin interactions, and developing antagonists for cell surface adhesion molecules, particularly the α_ν, β₃ vitronectin-like integrin receptor present on the surface of human osteoclasts. Peptides containing the internal arginine-glycine-aspartic acid (RGD) motif have been shown to inhibit osteoclast-mediated bone resorption in vivo. We are now designing more potent and selective inhibitors of bone resorption as a potential new mechanism-based therapeutic approach to osteoporosis based on a novel mechanism. In an effort to rapidly identify the highest affinity ligands for the human α_νβ₃ integrin, we have generated combinatorial peptide libraries containing substantial structural diversity. For instance, based on all possible sequence combinations of extracellular matrix proteins known to bind α_νβ₃, we recently synthesized and chemically analyzed a library of 360,000 peptides, all of which contain RGD. Human α_νβ₃ receptors are now available in a clonal cell line that expresses high levels of recombinant receptor, these cells can serve as a very important research tool in this project because of the limited number of bone-derived osteoclasts that can be harvested for experimentation. The library of peptides will be screened by “affinity selection”: the highest binding affinity peptide(s) will be isolated and microsequenced. Receptor-favored sequences will be synthesized and evaluated in a battery of in vitro and in vivo bioassays. Through these investigations, insight will be gained into the role of integrins in bone biology and patho-physiology, and new directions will be developed for the design of potent human α_νβ₃-selective antagonists for the treatment of osteoporosis. © 1994 John Wiley & Sons, Inc.     

Bumetanide-sensitive Na+ /K+ /Cl− transporter is stimulated by phorbol ester and different mitogens in quiescent human skin fibroblasts

In this study we investigated the correlation between the mitogenic effect and stimulation of Rb + (K + ) fluxes in human skin fibroblasts treated by purified growth factors. Both K+ transporters, bumetanide-sensitive and ouabain-sensitive, are stimulated 2-3-fold after addition of either fetal calf serum or purified recombinant growth factors to quiescent G₀/G₁ human skin fibroblasts. Three groups of mitogens were compared: (i) the phorbol ester 2-O-tetradecanoyl-phorbol-13-acetate (TPA); (ii) growth factors that stimulate inositol phosphate hydrolysis and subsequently activate protein kinase C—fibroblast growth factor (FGF), platelet derived growth factor (PDGF), and α-thrombin; and (iii) growth factors that do not activate kinase C—insulin-like growth factor-1 (IGF-1), and transforming like growth-factor-α (TGF-α). The three groups of mitogens stimulated human skin fibroblasts proliferation and Rb+ influxes in a similar dose-dependent fashion. The results indicate that both the bumetanide-sensitive and the ouabain-sensitive Rb+ fluxes are stimulated by protein kinase C-dependent and by the protein kinase C-independent pathways of the mitogenic signal.     

Hydrogen bond interactions of G proteins with the guanine ring moiety of guanine nucleotides.

We have utilized Raman difference spectroscopy to investigate hydrogen bonding interactions of the guanine moiety in guanine nucleotides with the binding site of two G proteins, EF-Tu (elongation factor Tu from Escherichia coli) and the c-Harvey ras protein, p21 (the gene product of the human c-H-ras proto-oncogene). Raman spectra of proteins complexed with GDP (guanosine 5' diphosphate), IDP (inosine 5' diphosphate), 6-thio-GDP, and 6-18O-GDP were measured, and the various difference spectra were determined. These were compared to the difference spectra obtained in solution, revealing vibrational features of the nucleotide that are altered upon binding. Specifically, we observed significant frequency shifts in the vibrational modes associated with the 6-keto and 2-amino positions of the guanine group of GDP and IDP that result from hydrogen bonding interactions between these groups and the two proteins. These shifts are interpreted as being proportional to the local energy of interaction (delta H) between the two groups and protein residues at the nucleotide binding site. Consistent with the tight binding between the nucleotides and the two proteins, the shifts indicate that the enthalpic interactions are stronger between these two polar groups and protein than with water. In general, the spectral shifts provide a rationale for the stronger binding of GDP and IDP with p21 compared to EF-Tu. Despite the structural similarity of the binding sites of EF-Tu and p21, the strengths of the observed hydrogen bonds at the 6-keto and 2-amino positions vary substantially, by up to a factor of 2.(ABSTRACT TRUNCATED AT 250 WORDS)