Biostructural Science Inspired by Next-Generation X-Ray Sources.

Next-generation synchrotron radiation sources, such as X-ray free-electron lasers, energy recovery linacs, and ultra-low-emittance storage rings, are catalyzing novel methods of biomolecular microcrystallography and solution scattering. These methods are described and future trends are predicted. Importantly, there is a growing realization that serial microcrystallography and certain cutting-edge solution scattering experiments can be performed at existing storage ring sources by utilizing new technology. In this sense, next-generation sources are serving two distinct functions, namely, provision of new capabilities that require the newer sources and inspiration of new methods that can be performed at existing sources.

West Nile virus encephalitis acquired via liver transplantation and clinical response to intravenous immunoglobulin: case report and review of the literature.

A patient developed West Nile virus (WNV) encephalitis 2 weeks after receiving a liver transplant and recovered fully, following treatment with intravenous immunoglobulin (IVIg). Laboratory testing documented transmission from the organ donor. Clinicians should be suspicious for organ-transmitted WNV in any post-transplant patient who develops fever and neurological symptoms. We review previous cases of organ-transmitted WNV, the use of IVIg for WNV encephalitis, and the issue of organ donor screening.

Metallothionein induces a regenerative reactive astrocyte phenotype via JAK/STAT and RhoA signalling pathways.

Following central nervous system injury, astrocytes rapidly respond by undergoing a stereotypical pattern of molecular and morphological alterations termed "reactive" astrogliosis. We have reported previously that metallothioneins (MTs) are rapidly expressed by reactive astrocytes and that their secretion and subsequent interaction with injured neurons leads to improved neuroregeneration. We now demonstrate that exogenous MT induces a reactive morphology and elevated GFAP expression in cultured astrocytes. Furthermore, these astrogliotic hallmarks were mediated via JAK/STAT and RhoA signalling pathways. However, rather than being inhibitory, MT induced a form of astrogliosis that was permissive to neurite outgrowth and which was associated with decreased chondroitin sulphate proteoglycan (CSPG) expression. The results suggest that MT has an important role in mediating permissive astrocytic responses to traumatic brain injury.

Metallothionein treatment attenuates microglial activation and expression of neurotoxic quinolinic acid following traumatic brain injury.

The kynurenine pathway has been implicated as a major component of the neuroinflammatory response to brain injury and neurodegeneration. We found that the neurotoxic kynurenine pathway intermediate quinolinic acid (QUIN) is rapidly expressed, within 24 h, by reactive microglia following traumatic injury to the rodent neocortex. Furthermore, administration of the astrocytic protein metallothionein attenuated this neuroinflammatory response by reducing microglial activation (by approximately 30%) and QUIN expression. The suppressive effect of MT was confirmed upon cultured cortical microglia, with 1 mug/ml MT almost completely blocking interferon-gamma induced activation of microglia and QUIN expression. These results demonstrate the neuroimmunomodulatory properties of MT, which may have therapeutic applications for the treatment of traumatic brain injury.

A method for finding candidate conformations for molecular replacement using relative rotation between domains of a known structure.

This paper presents a methodology to obtain candidate conformations of multidomain proteins for use in molecular replacement. For each separate domain, the orientational relationship between the template and the target structure is obtained using standard molecular replacement. The orientational relationships of the domains are then used to calculate the relative rotation between the domains in the target conformation by using pose-estimation techniques from the field of robotics and computer vision. With the angle of relative rotation between the domains as a cost function, iterative normal-mode analysis is used to drive the template structure to a candidate conformation that matches the X-ray crystallographic data obtained for the target conformation. The selection of the correct intra-protein domain orientations from among the many spurious maxima in the rotation function (including orientations obtained from domains in symmetry mates rather than within the same copy of the protein) presents a challenge. This problem is resolved by checking R factors of each domain, measuring the absolute value of relative rotation between domains, and evaluating the cost value after each candidate conformation is driven to convergence with iterative NMA. As a validation, the proposed method is applied to three test proteins: ribose-binding protein, lactoferrin and calcium ATPase. In each test case, the orientation and translation of the final candidate conformation in the unit cell are generated correctly from the suggested procedure. The results show that the proposed method can yield viable candidate conformations for use in molecular replacement and can reveal the structural details and pose of the target conformation in the crystallographic unit cell.

Molecular dynamics study of water penetration in staphylococcal nuclease.

The ionization properties of Lys and Glu residues buried in the hydrophobic core of staphylococcal nuclease (SN) suggest that the interior of this protein behaves as a highly polarizable medium with an apparent dielectric constant near 10. This has been rationalized previously in terms of localized conformational relaxation concomitant with the ionization of the internal residue, and with contributions by internal water molecules. Paradoxically, the crystal structure of the SN V66E variant shows internal water molecules and the structure of the V66K variant does not. To assess the structural and dynamical character of interior water molecules in SN, a series of 10-ns-long molecular dynamics (MD) simulations was performed with wild-type SN, and with the V66E and V66K variants with Glu66 and Lys66 in the neutral form. Internal water molecules were identified based on their coordination state and characterized in terms of their residence times, average location, dipole moment fluctuations, hydrogen bonding interactions, and interaction energies. The locations of the water molecules that have residence times of several nanoseconds and display small mean-square displacements agree well with the locations of crystallographically observed water molecules. Additional, relatively disordered water molecules that are not observed crystallographically were found in internal hydrophobic locations. All of the interior water molecules that were analyzed in detail displayed a distribution of interaction energies with higher mean value and narrower width than a bulk water molecule. This underscores the importance of protein dynamics for hydration of the protein interior. Further analysis of the MD trajectories revealed that the fluctuations in the protein structure (especially the loop elements) can strongly influence protein hydration by changing the patterns or strengths of hydrogen bonding interactions between water molecules and the protein. To investigate the dynamical response of the protein to burial of charged groups in the protein interior, MD simulations were performed with Glu66 and Lys66 in the charged state. Overall, the MD simulations suggest that a conformational change rather than internal water molecules is the dominant determinant of the high apparent polarizability of the protein interior.

Influence of ions, hydration, and the transcriptional inhibitor P4N on the conformations of the Sp1 binding site.

Three crystal structures containing the entire Sp1 consensus sequence d(GGGGCGGGG) with two or three additional base-pairs on either the 5' or 3' ends and overhangs have been determined. Despite the different lengths of DNA in the pseudo-dodecamers and pseudo-tridecamer, all three structures form A-DNA duplexes that share a common set of crystal contacts, including a T*(G.C) base triplet and a 5'-overhang that flips out and away from the helical axes to form a Hoogsteen base-pair with the 3'-overhang of a symmetry mate. The global conformations of the three structures differ, however, in the widths of their respective major grooves, the lengths of the molecules, and the extent of crystal packing. The structures were determined from crystals grown in an unusual precipitant for A-DNA, polyethylene glycol (PEG) 400, in combination with polyamines or ions; cobalt hexamine for the pseudo-tridecamer, and spermidine for the pseudo-dodecamers. As the Sp1 binding site is a target for antiviral and anticancer drugs, pseudo-dodecamer crystals were soaked with one such antiviral and anticancer compound, P4N. Although P4N was not visualized unambiguously in the electron density maps, the effect of the drug is evident from significant differences in the lattice constants, crystal packing, and overall conformation of the structure.

User-driven design of a computerized rounding and sign-out application.

Clinical information systems depend on close integration to workflow for success. We describe a method for user-driven design that guided our development of a computerized rounding and sign-out system. The resulting system supported clinical workflow sufficiently well that it spontaneously attracted new users, required no training, and is currently used by 95% of the house staff at two academic medical centers.

X-ray and thermodynamic studies of staphylococcal nuclease variants I92E and I92K: insights into polarity of the protein interior.

We have used crystallography and thermodynamic analysis to study nuclease variants I92E and I92K, in which an ionizable side-chain is placed in the hydrophobic core of nuclease. We find that the energetic cost of burying ionizable groups is rather modest. The X-ray determinations show water molecules solvating the buried glutamic acid under cryo conditions, but not at room temperature. The lysine side-chain does not appear solvated in either case. Guanidine hydrochloride (GnHCl) denaturation of I92E and I92K, done as a function of pH and monitored by tryptophan fluorescence, showed that I92E and I92K are folded in the pH range pH 3.5-9.0 and pH 5.5-9.5, respectively. The stability of the parental protein is independent of pH over a broad range. In contrast, the stabilities of I92E and I92K exhibit a pH dependence, which is quantitatively explained by thermodynamic analysis: the PK(a) value of the buried K92 is 5.6, while that of the buried E92 is 8.65. The free energy difference between burying the uncharged and charged forms of the groups is modest, about 6 kcal/mol. We also found that epsilon(app) for I92K and I92E is in the range approximately 10-12, instead of 2-4 commonly used to represent the protein interior. Side-chains 92E and 92K were uncharged under the conditions of the X-ray experiment. Both are buried completely inside the well-defined hydrophobic core of the variant proteins without forming salt-bridges or hydrogen bonds to other functional groups of the proteins. Under cryo conditions 92E shows a chain of four water molecules, which hydrate one oxygen atom of the carboxyl group of the glutamic acid. Two other water molecules, which are present in the wild-type at all temperatures, are also connected to the water ring observed inside the hydrophobic core. The ready burial of water with an uncharged E92 raises the possibility that solvent excursions into the interior also take place in the wild-type protein, but in a random, dynamic way not detectable by crystallography. Such transient excursions could increase the average polarity, and thus epsilon(app), of the protein interior.

A compact RNA tertiary structure contains a buried backbone-K+ complex.

The structure of a 58 nucleotide ribosomal RNA fragment buries several phosphate groups of a hairpin loop within a large tertiary core. During refinement of an X-ray crystal structure containing this RNA, a potassium ion was found to be contacted by six oxygen atoms from the buried phosphate groups; the ion is contained completely within the solvent-accessible surface of the RNA. The electrostatic potential at the ion chelation site is unusually large, and more than compensates for the substantial energetic penalties associated with partial dehydration of the ion and displacement of delocalized ions. The very large predicted binding free energy, approximately -30 kcal/mol, implies that the site must be occupied for the RNA to fold. These findings agree with previous studies of the ion-dependent folding of tertiary structure in this RNA, which concluded that a monovalent ion was bound in a partially dehydrated environment where Mg2+ could not easily compete for binding. By compensating the unfavorable free energy of buried phosphate groups with a chelated ion, the RNA is able to create a larger and more complex tertiary fold than would be possible otherwise.

Experimental pK(a) values of buried residues: analysis with continuum methods and role of water penetration.

Lys-66 and Glu-66, buried in the hydrophobic interior of staphylococcal nuclease by mutagenesis, titrate with pK(a) values of 5.7 and 8.8, respectively (Dwyer et al., Biophys. J. 79:1610-1620; García-Moreno E. et al., Biophys. Chem. 64:211-224). Continuum calculations with static structures reproduced the pK(a) values when the protein interior was treated with a dielectric constant (epsilon(in)) of 10. This high apparent polarizability can be rationalized in the case of Glu-66 in terms of internal water molecules, visible in crystallographic structures, hydrogen bonded to Glu-66. The water molecules are absent in structures with Lys-66; the high polarizability cannot be reconciled with the hydrophobic environment surrounding Lys-66. Equilibrium thermodynamic experiments showed that the Lys-66 mutant remained folded and native-like after ionization of the buried lysine. The high polarizability must therefore reflect water penetration, minor local structural rearrangement, or both. When in pK(a) calculations with continuum methods, the internal water molecules were treated explicitly, and allowed to relax in the field of the buried charged group, the pK(a) values of buried residues were reproduced with epsilon(in) in the range 4-5. The calculations show that internal waters can modulate pK(a) values of buried residues effectively, and they support the hypothesis that the buried Lys-66 is in contact with internal waters even though these are not seen crystallographically. When only the one or two innermost water molecules were treated explicitly, epsilon(in) of 5-7 reproduced the pK(a) values. These values of epsilon(in) > 4 imply that some conformational reorganization occurs concomitant with the ionization of the buried groups.

Cloning and characterization of murine p53 upstream sequences reveals additional positive transcriptional regulatory elements.

Transcriptional regulation of the p53 gene plays an important role leading to elevated expression of mutant p53 alleles in tumor cells. In addition, alterations in p53 transcription levels occur in response to changes in the cell cycle. Previous work had identified a number of regulatory sites at the 5'-end of the murine p53 promoter. During the characterization of the 5'-end of the cloned murine p53 promoter, we identified a 28 bp positive regulatory element that participates in three distinct DNA-protein complexes. The binding by nuclear factors to each one of these sites contributes to the overall activity of the p53 promoter. One site is a potential recognition sequence for members of the ETS family of transcription factors, which are known regulators of the human p53 promoter. Since six nucleotides in the middle of this required element were not present in the previously published sequence of the murine promoter, we recloned this region from C57/BL6 cells and confirmed their presence in the genome. The removal of this regulatory element completely abolishes p53 promoter activity.

hSIR2(SIRT1) functions as an NAD-dependent p53 deacetylase.

DNA damage-induced acetylation of p53 protein leads to its activation and either growth arrest or apoptosis. We show here that the protein product of the gene hSIR2(SIRT1), the human homolog of the S. cerevisiae Sir2 protein known to be involved in cell aging and in the response to DNA damage, binds and deacetylates the p53 protein with a specificity for its C-terminal Lys382 residue, modification of which has been implicated in the activation of p53 as a transcription factor. Expression of wild-type hSir2 in human cells reduces the transcriptional activity of p53. In contrast, expression of a catalytically inactive hSir2 protein potentiates p53-dependent apoptosis and radiosensitivity. We propose that hSir2 is involved in the regulation of p53 function via deacetylation.

Characterization of C/EBPbeta isoforms in normal versus neoplastic mammary epithelial cells.

A member of the CCAAT Enhancer Binding Proteins (C/EBPs) family of transcription factors, C/EBPbeta, has recently proven to be an important player in both growth and differentiation of the epithelial cells in the mammary gland. When the gene for C/EBPbeta is disrupted in mice, these mice fail to either develop normal mammary ducts during puberty or pregnancy, or to lactate upon parturition. C/EBPbeta can be present in cells in three isoforms: C/EBPbeta-1, -2, and -3. These isoforms have the same carboxy terminus but different N-termini due to alternative translational initiation at three different initiator codons within the C/EBPbeta mRNA. Using a commercially available antibody specific to the C-terminus of C/EBPbeta and a novel antibody specific to the N-terminus of C/EBPbeta-1, we have uncovered a striking difference in the forms of C/EBPbeta present in normal mammary epithelial cells versus breast cancer cell lines. C/EBPbeta- 1 is found exclusively in normal mammary epithelial cells, whereas C/EBPbeta- 2 is found only in dividing cells, both normal and neoplastic. Our preliminary data suggest that the prevalent form of C/EBPbeta in cancer cells, C/EBPbeta- 2, can activate genes which push the cell to divide, such as cyclin D1.

Interaction of the Ski oncoprotein with Smad3 regulates TGF-beta signaling.

TGF-beta treatment of cells induces a variety of physiologic responses, including growth inhibition, differentiation, and induction of apoptosis. TGF-beta induces phosphorylation and nuclear translocation of Smad3. We describe here the association of Smad3 with the nuclear protooncogene protein Ski in response to the activation of TGF-beta signaling. Association with Ski represses transcriptional activation by Smad3, and overexpression of Ski renders cells resistant to the growth-inhibitory effects of TGF-beta. The transcriptional repression as well as the growth resistance to TGF-beta by overexpression of Ski can be overcome by overexpression of Smad3. These results demonstrate that Ski is a novel component of the TGF-beta signaling pathway and shed light on the mechanism of action of the Ski oncoprotein.

SnoN and Ski protooncoproteins are rapidly degraded in response to transforming growth factor beta signaling.

Transforming growth factor beta (TGF-beta) regulates a variety of physiologic processes, including growth inhibition, differentiation, and induction of apoptosis. Some TGF-beta-initiated signals are conveyed through Smad3; TGF-beta binding to its receptors induces phosphorylation of Smad3, which then migrates to the nucleus where it functions as a transcription factor. We describe here the association of Smad3 with the nuclear protooncogene protein SnoN. Overexpression of SnoN represses transcriptional activation by Smad3. Activation of TGF-beta signaling leads to rapid degradation of SnoN and, to a lesser extent, of the related Ski protein, and this degradation is likely mediated by cellular proteasomes. These results demonstrate the existence of a cascade of the TGF-beta signaling pathway, which, upon TGF-beta stimulation, leads to the destruction of protooncoproteins that antagonize the activation of the TGF-beta signaling.

Inhibition of telomerase limits the growth of human cancer cells.

Telomerase is a ribonucleoprotein enzyme that maintains the protective structures at the ends of eukaryotic chromosomes, called telomeres. In most human somatic cells, telomerase expression is repressed, and telomeres shorten progressively with each cell division. In contrast, most human tumors express telomerase, resulting in stabilized telomere length. These observations indicate that telomere maintenance is essential to the proliferation of tumor cells. We show here that expression of a mutant catalytic subunit of human telomerase results in complete inhibition of telomerase activity, reduction in telomere length and death of tumor cells. Moreover, expression of this mutant telomerase eliminated tumorigenicity in vivo. These observations demonstrate that disruption of telomere maintenance limits cellular lifespan in human cancer cells, thus validating human telomerase reverse transcriptase as an important target for the development of anti-neoplastic therapies.