Studies of the ankyrin repeats of the Drosophila melanogaster Notch receptor. 1. Solution conformational and hydrodynamic properties.

To gain insight into the structural basis for Notch signaling, and to investigate the relationship between structure and stability in ankyrin repeat proteins, we have examined structural features of polypeptides from the Drosophila melanogaster Notch protein that contain five, six, and a putative seventh ankyrin repeat. Circular dichroism (CD) spectroscopy indicates that Notch ankyrin polypeptides of different length contain a significant amount of alpha-helix, indicating that a folded structure can be maintained despite the loss of individual ankyrin modules. However, the alpha-helical content of the construct with the putative seventh repeat is slightly higher than polypeptides containing fewer repeats, suggesting that the putative seventh repeat may help stabilize other parts of the ankyrin domain. Fluorescence spectroscopy indicates that the single tryptophan in the fifth ankyrin repeat is in a nonpolar environment and is shielded from solvent in all three constructs, although slight differences in spectroscopic properties of the six- and five-repeat constructs are observed, indicating minor structural changes. Near-UV CD indicates that these ankyrin polypeptides contain a significant amount of fixed tertiary structure surrounding their aromatic side chains. Gel filtration chromatography and sedimentation equilibrium studies indicate that these ankyrin repeat polypeptides are monomeric. Sedimentation velocity studies indicate that each polypeptide exhibits significant axial asymmetry, consistent with the elongated structure seen for other for ankyrin repeat proteins. However, the degree of asymmetry is greatest for the construct containing six repeats, suggesting that in the absence of the putative seventh repeat, the sixth repeat is partly unfolded.     

Studies of the ankyrin repeats of the Drosophila melanogaster Notch receptor. 2. Solution stability and cooperativity of unfolding.

To define the boundaries of the Drosophila Notch ankyrin domain, examine the effects of repeat number on the folding of this domain, and examine the degree to which the modular architecture of ankyrin repeat proteins results in modular stability, we have investigated the thermodynamics of unfolding of polypeptides corresponding to different segments of the ankyrin repeats of Drosophila Notch. We find that a polypeptide containing the six previously identified ankyrin repeats unfolds cooperatively, but is of modest stability. However, inclusion of a putative seventh, C-terminal ankyrin sequence doubles the stability of the Notch ankyrin domain (a 1000-fold increase in the folding equilibrium constant), indicating that the seventh ankyrin repeat is an important part of the Notch ankyrin domain, and demonstrating long-range interactions among ankyrin repeats. This putative seven-repeat polypeptide also shows increases in enthalpy, denaturant dependence (m-value), and heat capacity of unfolding (DeltaC(p)()) of around 50% each, suggesting that deletion of the seventh repeat results in partial unfolding of the sixth ankyrin repeat, consistent with spectroscopic and hydrodynamic data reported in the preceding paper [Zweifel, M. E., and Barrick, D. (2001) Biochemistry 40, 14344-14356]. A polypeptide consisting of only the five N-terminal repeats has stability similar to the six-repeat construct, demonstrating that stability is distributed asymmetrically along the ankyrin domain. These data are consistent with highly cooperative two-state folding of these ankyrin polypeptides, despite their modular architecture.     

Influence of Ileo-Caecal Cannulation and Oxytetracycline on Ileo-Caecal and Rectal Coliform Populations in Pigs

The effect of surgery (insertion of an ileo-caecal cannula) and a subsequent parenteral treatment with oxytetracycline on the ileo-caecal and rectal coliform populations in 7 Swedish Yorkshire castrates were studied. Samples were collected during surgery as well as 3, 7, 14 and 20 days post surgery. The diversity of the enteric coliform flora was initially high both in the ileo-caecal ostium and in rectum. No alteration in the diversity of the enteric coliform flora was observed following surgery and treatment with oxytetracycline. As the insertion of ileo-caecal cannulas did not affect the intestinal coliform flora this study gives support to the use of this technique to mirror processes in the small intestine of pigs. Further, the diversity of the enteric coliform flora was unaffected by the parenteral treatment with oxytetracycline.     

Distal ligand reactivity and quaternary structure studies of proximally detached hemoglobins

The linkage between the proximal histidines and the proximal polypeptide in normal adult human hemoglobin (Hb A) has been proposed to play a major role in transmitting allosteric effects between oxygen binding sites [Perutz, M. F. (1970) Nature 228, 726-734]. Here we present circular dichroism (CD), (1)H NMR, analytical ultracentrifugation, and stopped-flow kinetic data to better define the quaternary structure of hemoglobins in which the linkage between the proximal histidines and the polypeptide backbone has been broken [Barrick et al. Nat. Struct. Biol. 4, 78-83 (1997)] and to characterize the distal ligand binding properties of these proximally detached Hbs. CD spectroscopy indicates that rHb (alphaH87G) and rHb (alphaH87G/betaH92G) retain at least partial T-quaternary structure with distal ligand bound, whereas rHb (betaH92G) does not, consistent with (1)H NMR spectra. Analytical ultracentrifugation reveals significant tetramer dissociation in rHb (betaH92G) to be the likely cause of loss of T-state markers. These quaternary structure studies indicate that in distally liganded Hb, the T-state is compatible with proximal linkages in the beta- but not the alpha-chains. (1)H NMR titrations of rHb (alphaH87G) with n-butyl isocyanide demonstrate the alpha-chains to be of high affinity as compared with the beta-chains. Comparing ligand association and dissociation rates between the rHb (alphaH87G) variant with the T- and R-states of wild-type Hb A indicates that at the alpha-chains, carbon monoxide affinity is modulated entirely by the proximal linkage, rather than from distal interactions. Some residual allosteric interactions may remain operative at the beta-chains of rHb (alphaH87G).     

Structural maintenance of chromosomes (SMC) proteins, a family of conserved ATPases

The structural maintenance of chromosomes (SMC) proteins are essential for successful chromosome transmission during replication and segregation of the genome in all organisms. SMCs are generally present as single proteins in bacteria, and as at least six distinct proteins in eukaryotes. The proteins range in size from approximately 110 to 170 kDa, and each has five distinct domains: amino- and carboxy-terminal globular domains, which contain sequences characteristic of ATPases, two coiled-coil regions separating the terminal domains and a central flexible hinge. SMC proteins function together with other proteins in a range of chromosomal transactions, including chromosome condensation, sister-chromatid cohesion, recombination, DNA repair and epigenetic silencing of gene expression. Recent studies are beginning to decipher molecular details of how these processes are carried out.     

Measuring the stability of partly folded proteins using TMAO

Standard methods for measuring free energy of protein unfolding by chemical denaturation require complete folding at low concentrations of denaturant so that a native baseline can be observed. Alternatively, proteins that are completely unfolded in the absence of denaturant can be folded by addition of the osmolyte trimethylamine N-oxide (TMAO), and the unfolding free energy can then be calculated through analysis of the refolding transition. However, neither chemical denaturation nor osmolyte-induced refolding alone is sufficient to yield accurate thermodynamic unfolding parameters for partly folded proteins, because neither method produces both native and denatured baselines in a single transition. Here we combine urea denaturation and TMAO stabilization as a means to bring about baseline-resolved structural transitions in partly folded proteins. For Barnase and the Notch ankyrin domain, which both show two-state equilibrium unfolding, we found that DeltaG degrees for unfolding depends linearly on TMAO concentration, and that the sensitivity of DeltaG degrees to urea (the m-value) is TMAO independent. This second observation confirms that urea and TMAO exert independent effects on stability over the range of cosolvent concentrations required to bring about baseline-resolved structural transitions. Thermodynamic parameters calculated using a global fit that assumes additive, linear dependence of DeltaG degrees on each cosolvent are similar to those obtained by standard urea-induced unfolding in the absence of TMAO. Finally, we demonstrate the applicability of this method to measurement of the free energy of unfolding of a partly folded protein, a fragment of the full-length Notch ankyrin domain.     

Phosphorylation of Ezrin-Radixin-Moesin-binding Phosphoprotein 50 (EBP50) by Akt Promotes Stability and Mitogenic Function of S-phase Kinase-associated Protein-2 (Skp2)

The regulation of the cell cycle by the ubiquitin-proteasome system is dependent on the activity of E3 ligases. Skp2 (S-phase kinase associated protein-2) is the substrate recognition subunit of the E3 ligase that ubiquitylates the cell cycle inhibitors p21^cip1 and p27^kip1 thus promoting cell cycle progression. Increased expression of Skp2 is frequently observed in diseases characterized by excessive cell proliferation, such as cancer and neointima hyperplasia. The stability and cellular localization of Skp2 are regulated by Akt, but the molecular mechanisms underlying these effects remain only partly understood. The scaffolding protein Ezrin-Binding Phosphoprotein of 50 kDa (EBP50) contains two PDZ domains and plays a critical role in the development of neointimal hyperplasia. Here we report that EBP50 directly binds Skp2 via its first PDZ domain. Moreover, EBP50 is phosphorylated by Akt on Thr-156 within the second PDZ domain, an event that allosterically promotes binding to Skp2. The interaction with EBP50 causes cytoplasmic localization of Skp2, increases Skp2 stability and promotes proliferation of primary vascular smooth muscle cells. Collectively, these studies define a novel regulatory mechanism contributing to aberrant cell growth and highlight the importance of scaffolding function of EBP50 in Akt-dependent cell proliferation.     

Hormonally regulated programmed cell death in barley aleurone cells

Cell death was studied in barley (cv Himalaya) aleurone cells treated with abscisic acid and gibberellin. Aleurone protoplasts incubated in abscisic acid remained viable in culture for at least 3 weeks, but exposure to gibberellin initiated a series of events that resulted in death. Between 4 and 8 days after incubation in gibberellin, >70% of all protoplasts died. Death, which occurred after cells became highly vacuolated, was manifest by an abrupt loss of plasma membrane integrity followed by rapid shrinkage of the cell corpse. Hydrolysis of DNA began before death and occurred as protoplasts ceased production of alpha-amylase. DNA degradation did not result in the accumulation of discrete low molecular weight fragments. DNA degradation and cell death were prevented by LY83583, an inhibitor of gibberellin signaling in barley aleurone. We conclude that cell death in aleurone cells is hormonally regulated and is the final step of a developmental program that promotes successful seedling establishment.     

Mechanisms of Cognitive Impairment in Cerebral Small Vessel Disease: Multimodal MRI Results from the St George's Cognition and Neuroimaging in Stroke (SCANS) Study

Cerebral small vessel disease (SVD) is a common cause of vascular cognitive impairment. A number of disease features can be assessed on MRI including lacunar infarcts, T2 lesion volume, brain atrophy, and cerebral microbleeds. In addition, diffusion tensor imaging (DTI) is sensitive to disruption of white matter ultrastructure, and recently it has been suggested that additional information on the pattern of damage may be obtained from axial diffusivity, a proposed marker of axonal damage, and radial diffusivity, an indicator of demyelination. We determined the contribution of these whole brain MRI markers to cognitive impairment in SVD. Consecutive patients with lacunar stroke and confluent leukoaraiosis were recruited into the ongoing SCANS study of cognitive impairment in SVD (n = 115), and underwent neuropsychological assessment and multimodal MRI. SVD subjects displayed poor performance on tests of executive function and processing speed. In the SVD group brain volume was lower, white matter hyperintensity volume higher and all diffusion characteristics differed significantly from control subjects (n = 50). On multi-predictor analysis independent predictors of executive function in SVD were lacunar infarct count and diffusivity of normal appearing white matter on DTI. Independent predictors of processing speed were lacunar infarct count and brain atrophy. Radial diffusivity was a stronger DTI predictor than axial diffusivity, suggesting ischaemic demyelination, seen neuropathologically in SVD, may be an important predictor of cognitive impairment in SVD. Our study provides information on the mechanism of cognitive impairment in SVD.