The MntC crystal structure suggests that import of Mn2+ in cyanobacteria is redox controlled

The MntC protein is the periplasmic solute-binding protein component of the high-affinity manganese ATP-binding cassette-type transport system in the cyanobacterium Synechocytis PCC sp. 6803. We have determined the structure of recombinant MntC at 2.9 A resolution by X-ray crystallography using a combination of multi-wavelength anomalous diffraction and molecular replacement. The presence of Mn2+ in the metal ion-binding site was ascertained by use of anomalous difference electron density maps using diffraction data collected at the Mn absorption edge. The MntC protein is similar to previously determined metal ion-binding, solute-binding proteins with two globular domains connected by an extended alpha-helix. However, the metal ion-binding site is asymmetric, with two of the four ligating residues (Glu220 and Asp295) situated closer to the ion than the two histidine residues (His89 and His154). A unique characteristic of the MntC is the existence of a disulfide bond between Cys219 and Cys268. Analysis of amino acid sequences of homologous proteins shows that conservation of the cysteine residues forming the disulfide bond occurs only in cyanobacterial manganese solute-binding proteins. One of the monomers in the MntC asymmetric unit trimer is disordered significantly in the globular domain containing the disulfide bond. The electron density on the manganese ion and on the disulfide bond in this monomer indicates that reduction of this bond changes the relative position of the lower domain and of the Glu220 ligand, potentially lowering the affinity towards Mn2+. This is confirmed by reduction of the disulfide bond in vitro, showing the release of bound Mn2+. We propose that the reduction or oxidation state of the disulfide bond can alter the binding affinity of the protein towards Mn2+ and thus determine whether these ions will be transported into the cytoplasm, or be available for photosystem II biogenesis in the periplasm.     

Brain mechanisms for extracting spatial information from smell

Forty years ago, von Békésy demonstrated that the spatial source of an odorant is determined by comparing input across nostrils, but it is unknown how this comparison is effected in the brain. To address this, we delivered odorants to the left or right of the nose, and contrasted olfactory left versus right localization with olfactory identification during brain imaging. We found nostril-specific responses in primary olfactory cortex that were predictive of the accuracy of left versus right localization, thus providing a neural substrate for the behavior described by von Békésy. Additionally, left versus right localization preferentially engaged a portion of the superior temporal gyrus previously implicated in visual and auditory localization, suggesting that localization information extracted from smell was then processed in a convergent brain system for spatial representation of multisensory inputs.     

The phosphorylation status of the serine-rich region of the human cytomegalovirus 86-kilodalton major immediate-early protein IE2/IEP86 affects temporal viral gene expression

The 86-kDa major immediate-early protein (IE2/IEP86) of human cytomegalovirus (HCMV) contains a serine-rich region (amino acids 258 to 275) with several consensus casein kinase II (CKII) sites. We performed extensive mutational analysis of this region, changing serines to alternating alanines and glycines. Mutation of the serines between amino acids 266 and 275 eliminated in vitro phosphorylation by CKII. In vitro CKII phosphorylation of the serines between amino acids 266 and 269 or between amino acids 271 and 275 inhibited the ability of IE2/IEP86 to bind to TATA-binding protein. Correspondingly, nonphosphorylatable mutants in these regions showed increased activation of specific HCMV gene promoters in transfection studies. Viruses containing mutations of the serines throughout the entire region (amino acids 258 to 275) or the second half (amino acids 266 to 275) of the region showed delayed expression of all viral proteins tested and, correspondingly, delayed growth compared to wild-type HCMV. Mutation of the serines in the first half of the serine-rich region (amino acids 258 to 264) or between amino acids 266 and 269 propagated very slowly and has not been further studied. In contrast, mutation of the serines between amino acids 271 and 275 resulted in accelerated virus growth and accelerated temporal expression of viral proteins. These results suggest that the serine-rich region is structurally complex, possibly affecting multiple functions of IE2/IEP86. The data show that the phosphorylation state of the serine-rich region, particularly between amino acids 271 and 275, modulates the temporal expression of viral genes.     

The CD85J/leukocyte inhibitory receptor-1 distinguishes between conformed and beta 2-microglobulin-free HLA-G molecules

For a proper development of the placenta, maternal NK cells should not attack the fetal extravillous cytotrophoblast cells. This inhibition of maternal NK cells is partially mediated via the nonclassical MHC class I molecule HLA-G. Recently, we demonstrated that HLA-G forms disulfide-linked high molecular complexes on the surface of transfected cells. In the present study, we demonstrate that HLA-G must associate with beta(2)m for its interaction with CD85J/leukocyte Ig-like receptor-1 (LIR-1). Although HLA-G free H chain complexes are expressed on the surface, they are not recognized and possibly interfere with CD85J/LIR-1 and HLA-G interaction. The formation of these complexes on the cell surface might represent a novel mechanism developed specifically by the HLA-G protein aimed to control the efficiency of the CD85J/LIR-1-mediated inhibition. We also show that endogenous HLA-G complexes are expressed on the cell surface. These findings provide novel insights into the delicate interaction between extravillous cytotrophoblast cells and NK cells in the decidua.     

Refined structure of c-phycocyanin from the cyanobacterium Synechococcus vulcanus at 1.6 A: insights into the role of solvent molecules in thermal stability and co-factor structure

The crystal structure of the light-harvesting phycobiliprotein, c-phycocyanin from the thermophilic cyanobacterium Synechococcus vulcanus has been refined to 1.6 A resolution based on the previously determined lower resolution structure (PDB entry 1I7Y). The improved data was collected using synchrotron radiation at 100 K. The significantly improved crystallographic data has lead to improved calculated electron density maps, allowing the unambiguous positioning of all protein and co-factor atoms and the positioning of 377 solvent molecules. The positions of solvent molecules at specific sites important for stabilization of different levels of self-assembly of the phycobilisome structure were identified and the bonding network is described. The presence of solvent molecules in the vicinity of the co-factors and in intermolecular spaces is identified and their possible roles are suggested. All three of the phycocyanobilin co-factors bind water molecules at specific sites between the propionic acid side chains. Molecular dynamic (MD) simulations support that these special waters have a role in stabilization of this conformation. On the basis of the crystal packing reported here and in comparison to other phycobiliprotein crystal forms, we have analyzed the roles of specific sites on the formation of the phycobilisome complex.     

Deregulation of oncogene‐induced senescence and p53 translational control in X‐linked dyskeratosis congenita

Defects in ribosome biogenesis and function are present in a growing list of human syndromes associated with cancer susceptibility. One example is X‐linked dyskeratosis congenita (X‐DC) in which the DKC1 gene, encoding for an enzyme that modifies ribosomal RNA, is found to be mutated. How ribosome dysfunction leads to cancer remains poorly understood. A critical cellular response that counteracts cellular transformation is oncogene‐induced senescence (OIS). Here, we show that during OIS, a switch between cap‐ and internal ribosome entry site (IRES)‐dependent translation occurs. During this switch, an IRES element positioned in the 5′untranslated region of p53 is engaged and facilitates p53 translation. We further show that in DKC1^m cells, p53 IRES‐dependent translation is impaired during OIS ex vivo and on DNA damage in vivo. This defect in p53 translation perturbs the cellular response that counteracts oncogenic insult. We extend these findings to X‐DC human patient cells in which similar impairments in p53 IRES‐dependent translation are observed. Importantly, re‐introduction of wild‐type DKC1 restores p53 expression in these cells. These results provide insight into the basis for cancer susceptibility in human syndromes associated with ribosome dysfunction.     

A Self-Directed Method for Cell-Type Identification and Separation of Gene Expression Microarrays

Gene expression analysis is generally performed on heterogeneous tissue samples consisting of multiple cell types. Current methods developed to separate heterogeneous gene expression rely on prior knowledge of the cell-type composition and/or signatures - these are not available in most public datasets. We present a novel method to identify the cell-type composition, signatures and proportions per sample without need for a-priori information. The method was successfully tested on controlled and semi-controlled datasets and performed as accurately as current methods that do require additional information. As such, this method enables the analysis of cell-type specific gene expression using existing large pools of publically available microarray datasets.     

In Vivo 7T MRI of the Non-Human Primate Brainstem

Structural brain imaging provides a critical framework for performing stereotactic and intraoperative MRI-guided surgical procedures, with procedural efficacy often dependent upon visualization of the target with which to operate. Here, we describe tools for in vivo, subject-specific visualization and demarcation of regions within the brainstem. High-field 7T susceptibility-weighted imaging and diffusion-weighted imaging of the brain were collected using a customized head coil from eight rhesus macaques. Fiber tracts including the superior cerebellar peduncle, medial lemniscus, and lateral lemniscus were identified using high-resolution probabilistic diffusion tractography, which resulted in three-dimensional fiber tract reconstructions that were comparable to those extracted from sequential application of a two-dimensional nonlinear brain atlas warping algorithm. In the susceptibility-weighted imaging, white matter tracts within the brainstem were also identified as hypointense regions, and the degree of hypointensity was age-dependent. This combination of imaging modalities also enabled identifying the location and extent of several brainstem nuclei, including the periaqueductal gray, pedunculopontine nucleus, and inferior colliculus. These clinically-relevant high-field imaging approaches have potential to enable more accurate and comprehensive subject-specific visualization of the brainstem and to ultimately improve patient-specific neurosurgical targeting procedures, including deep brain stimulation lead implantation.