Human DNA-activated protein kinase phosphorylates serines 15 and 37 in the amino-terminal transactivation domain of human p53.

Human DNA-PK is a nuclear, serine/threonine protein kinase that, when activated by DNA, phosphorylates several DNA-binding substrates, including the tumor suppressor protein p53. To identify which p53 residues are phosphorylated, we examined DNA-PK's ability to phosphorylate synthetic peptides corresponding to human p53 sequences. Serines 15 and 37 in the amino-terminal transactivation domain of human p53, and serines 7 and 18 of mouse p53, were phosphorylated by DNA-PK in the context of synthetic peptides. Other serines in these p53 peptides, and serines in other p53 peptides, including peptides containing the serine 315 p34cdc2 site and the serine 392 casein kinase II site, were not recognized by DNA-PK or were phosphorylated less efficiently. Phosphorylation of the conserved serine 15 in human p53 peptides depended on the presence of an adjacent glutamine, and phosphorylation was inhibited by the presence of a nearby lysine. Phosphorylation of recombinant wild-type mouse p53 was inhibited at high DNA concentrations, suggesting that DNA-PK may phosphorylate p53 only when both are bound to DNA at nearby sites. Our study suggests that DNA-PK may have a role in regulating cell growth and indicates how phosphorylation of serine 15 in DNA-bound p53 could alter p53 function.     

Molecular biodiversity of cassava begomoviruses in Tanzania: evolution of cassava geminiviruses in Africa and evidence for East Africa being a center of diversity of cassava geminiviruses

Cassava is infected by numerous geminiviruses in Africa and India that cause devastating losses to poor farmers. We here describe the molecular diversity of seven representative cassava mosaic geminiviruses (CMGs) infecting cassava from multiple locations in Tanzania. We report for the first time the presence of two isolates in East Africa: (EACMCV-[TZ1] and EACMCV-[TZ7]) of the species East African cassava mosaic Cameroon virus, originally described in West Africa. The complete nucleotide sequence of EACMCV-[TZ1] DNA-A and DNA-B components shared a high overall sequence identity to EACMCV-[CM] components (92% and 84%). The EACMCV-[TZ1] and -[TZ7] genomic components have recombinations in the same genome regions reported in EACMCV-[CM], but they also have additional recombinations in both components. Evidence from sequence analysis suggests that the two strains have the same ancient origin and are not recent introductions. EACMCV-[TZ1] occurred widely in the southern part of the country. Four other CMG isolates were identified: two were close to the EACMV-Kenya strain (named EACMV-[KE/TZT] and EACMV-[KE/TZM] with 96% sequence identity); one isolate, TZ10, had 98% homology to EACMV-UG2Svr and was named EACMV-UG2 [TZ10]; and finally one isolate was 95% identical to EACMV-[TZ] and named EACMV-[TZ/YV]. One isolate of African cassava mosaic virus with 97% sequence identity with other isolates of ACMV was named ACMV-[TZ]. It represents the first ACMV isolate from Tanzania to be sequenced. The molecular variability of CMGs was also evaluated using partial B component nucleotide sequences of 13 EACMV isolates from Tanzania. Using the sequences of all CMGs currently available, we have shown the presence of a number of putative recombination fragments that are more prominent in all components of EACMV than in ACMV. This new knowledge about the molecular CMG diversity in East Africa, and in Tanzania in particular, has led us to hypothesize about the probable importance of this part of Africa as a source of diversity and evolutionary change both during the early stages of the relationship between CMGs and cassava and in more recent times. The existence of multiple CMG isolates with high DNA genome diversity in Tanzania and the molecular forces behind this diversity pose a threat to cassava production throughout the African continent.     

Utilization of oriented peptide libraries to identify substrate motifs selected by ATM

The ataxia telangiectasia mutated (ATM) gene encodes a serine/threonine protein kinase that plays a critical role in genomic surveillance and development. Here, we use a peptide library approach to define the in vitro substrate specificity of ATM kinase activity. The peptide library analysis identified an optimal sequence with a central core motif of LSQE that is preferentially phosphorylated by ATM. The contributions of the amino acids surrounding serine in the LSQE motif were assessed by utilizing specific peptide libraries or individual peptide substrates. All amino acids comprising the LSQE sequence were critical for maximum peptide substrate suitability for ATM. The DNA-dependent protein kinase (DNA-PK), a Ser/Thr kinase related to ATM and important in DNA repair, was compared with ATM in terms of peptide substrate selectivity. DNA-PK was found to be unique in its preference of neighboring amino acids to the phosphorylated serine. Peptide library analyses defined a preferred amino acid motif for ATM that permits clear distinctions between ATM and DNA-PK kinase activity. Data base searches using the library-derived ATM sequence identified previously characterized substrates of ATM, as well as novel candidate substrate targets that may function downstream in ATM-directed signaling pathways.     

Neonatal androgenization of hypogonadal (hpg) male mice does not abolish estradiol-induced FSH production and spermatogenesis

Background  

Testicular development is arrested in the hypogonadal (hpg) mouse due to a congenital deficiency in hypothalamic gonadotropin-releasing hormone (GnRH) synthesis. Chronic treatment of male hpg mice with estradiol induces FSH synthesis and secretion, and causes testicular maturation and qualitatively normal spermatogenesis. As estradiol negative feedback normally inhibits FSH production in the male, this study tested whether this paradoxical response to estradiol in the male hpg mouse might be due to inadequate masculinisation or incomplete defeminization in the neonatal period. Previous studies have demonstrated that treatment of hpg mice with testosterone propionate in the immediate neonatal period is necessary to allow full reproductive behaviors to be expressed following suitable endocrine stimulation at adult ages.     

XRCC4 protein interactions with XRCC4-like factor (XLF) create an extended grooved scaffold for DNA ligation and double strand break repair

The XRCC4-like factor (XLF)-XRCC4 complex is essential for nonhomologous end joining, the major repair pathway for DNA double strand breaks in human cells. Yet, how XLF binds XRCC4 and impacts nonhomologous end joining functions has been enigmatic. Here, we report the XLF-XRCC4 complex crystal structure in combination with biophysical and mutational analyses to define the XLF-XRCC4 interactions. Crystal and solution structures plus mutations characterize alternating XRCC4 and XLF head domain interfaces forming parallel super-helical filaments. XLF Leu-115 ("Leu-lock") inserts into a hydrophobic pocket formed by XRCC4 Met-59, Met-61, Lys-65, Lys-99, Phe-106, and Leu-108 in synergy with pseudo-symmetric β-zipper hydrogen bonds to drive specificity. XLF C terminus and DNA enhance parallel filament formation. Super-helical XLF-XRCC4 filaments form a positively charged channel to bind DNA and align ends for efficient ligation. Collective results reveal how human XLF and XRCC4 interact to bind DNA, suggest consequences of patient mutations, and support a unified molecular mechanism for XLF-XRCC4 stimulation of DNA ligation.     

Isolation and characterization of an abundant and novel 22-kDa protein (SM22) from chicken gizzard smooth muscle

Chicken gizzard smooth muscle contains a highly abundant protein (SM22) with an apparent Mr on sodium dodecyl sulfate-polyacrylamide electrophoretic gels of 23,000. The ratio of actin:SM22:tropomyosin in this tissue is estimated to be 6.5(+/- 0.8):2.0(+/- 0.2):1.0. At least three isoelectric isoforms are present in ratios of alpha:beta:gamma of 14:5:1 with alpha the most basic and gamma the most acidic. A method for the purification of SM22 and partial separation of its isoforms is described. Amino acid analyses of purified alpha and beta demonstrate the presence of 1 and 2 half-cystines, respectively, and a lower content of basic amino acids in beta. A value of 22,000 for the Mr of alpha estimated by sedimentation equilibrium indicated its presence as a monomer at physiological ionic strengths. Estimates of the translational frictional coefficient (f/fmin) of alpha calculated from its Stokes radius (25.5 A) and Mr were consistent with its existence as a moderately asymmetric globular protein. Calculations based on its far-ultraviolet CD spectrum provided values of 37% alpha-helix, 31% beta-sheet, 5% beta-turn, and 27% random coil. SM22 was shown not to share functional properties with several proteins of similar Mr and isoelectric point such as myokinase, brain 23-kDa protein, and troponin I. We conclude that it is a novel protein not previously isolated or characterized from any tissue.     

Biochemical characterization of the ataxia-telangiectasia mutated (ATM) protein from human cells

Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in controlling the cellular response to ionizing radiation and other DNA-damaging agents. ATM is a 3056 amino acid polypeptide that is present in low abundance in the nucleus of human cells. Here, we describe the purification and characterization of ATM from the nuclear fraction of HeLa cells. Microgram quantities of highly stable, kinase-active ATM were prepared. Purified ATM was phosphorylated on serine 1981 and was active towards a variety of known ATM substrates, including p53 and the Bloom Syndrome helicase, BLM. The protein kinase activity of ATM was selectively inhibited by wortmannin, caffeine and LY294002 and was stimulated by charged biological polymers, including single-stranded M13 DNA (ssDNA), sheared double-stranded calf thymus DNA, heparin sulfate and poly ADP-ribose (PAR), raising the possibility that charged structures may contribute to regulation of ATM activity. However, chemical inhibition of the formation of poly ADP-ribose in cells had no effect on the activation of ATM-dependent pathways by ionizing radiation. Using gel filtration chromatography, we also show that purified ATM, as well as ATM in crude nuclear extracts from unirradiated and irradiated cells elutes with an estimated native molecular weight of approximately 600 kDa. Moreover, dephosphorylation of serine 1981 did not affect the apparent molecular weight of ATM in irradiated extracts. Our results suggest that phosphorylation of serine 1981 alone may not directly regulate the subunit composition of ATM.     

Decrease in density of INa is in the common final pathway to heart block in murine hearts overexpressing calcineurin

Overexpression of calcineurin in transgenic mouse heart results in massive cardiac hypertrophy followed by sudden death. Sudden deaths are caused by abrupt transitions from sinus rhythm to heart block (asystole) in calcineurin-overexpressing (CN) mice. Preliminary studies showed decreased maximum change in potential over time (dV/dt(max)) of phase 0 of the action potential. Accordingly, the hypothesis was tested that decreased activity of the sodium channel contributes to heart block. Profound decreases in activity of sodium currents (I(Na)) paralleled the changes in action potential characteristics. Progressive age-dependent decreases were observed such that at 42-50 days of life little sodium channel function existed. However, this was not paralleled by decreased protein expression as assessed by immunocytochemistry or by Western blot. Since calcineurin can interact with the ryanodine receptor, we assessed whether chronic in vitro treatment with BAPTA-AM, thapsigargin, and ryanodine could rescue the decrease of I(Na). All of these treatments rescued I(Na) to levels indistinguishable from wild type. The nonspecific PKC inhibitor bisindolylmaleimide I also rescued the decrease of I(Na). To assess whether decreased sodium channel activity contributes to sudden death in vivo, the response to encainide (20 mg/kg) was assessed: 6 of 10 young CN mice died because of asystole, whereas 0 of 10 wild-type mice died (P < 0.01). Moreover, encainide produced exaggerated prolongation of the QRS width in sinus beats before the heart block. Catecholamine tone appears necessary to support life in older CN mice because propranolol (1 mg/kg) triggered asystolic death in five of six CN mice. We conclude that decrease in sodium channel activity is in the common final pathway to asystole in CN mice.     

Immune risk phenotype is associated with nosocomial lung infections in elderly in-patients

Background

Nosocomial infections are extremely common in the elderly and may be related to ageing of the immune system. The Immune Risk Phenotype (IRP), which predicts shorter survival in elderly patients, has not been evaluated as a possible risk factor for nosocomial infection. Our aim was to assess the prevalence of nosocomial infections in elderly in-patients and to investigate potential relationships between nosocomial infections and the immunophenotype, including IRP parameters.

Results

We included 252 consecutive in-patients aged 70 years or over (mean age, 85 ± 6.2 years), between 2006 and 2008. Among them, 97 experienced nosocomial infections, yielding a prevalence rate of 38.5% (95% confidence interval, 32.5-44.5). The main infection sites were the respiratory tract (21%) and urinary tract (17.1%) When we compared immunological parameters including cell counts determined by flow cytometry in the groups with and without nosocomial infections, we found that the group with nosocomial infections had significantly lower values for the CD4/CD8 ratio and naive CD8 and CD4 T-cell counts and higher counts of memory CD8 T-cells with a significant increase in CD28-negative CD8-T cells. Neither cytomegalovirus status (positive in 193/246 patients) nor presence of the IRP was associated with nosocomial infections. However, nosocomial pneumonia was significantly more common among IRP-positive patients than IRP-negative patients (17/60 versus 28/180; p = 0.036).

Conclusion

Immunological parameters that are easy to determine in everyday practice and known to be associated with immune system ageing and shorter survival in the elderly are also associated with an elevated risk of nosocomial pneumonia in the relatively short term.