Three-Dimensional X-ray Imaging of ß-Galactosidase Reporter Activity by Micro-CT: Implication for Quantitative Analysis of Gene Expression.

Acquisition of detailed anatomical and molecular knowledge from intact biological samples while preserving their native three-dimensional structure is still a challenging issue for imaging studies aiming to unravel a system's functions. Three-dimensional micro-CT X-ray imaging with a high spatial resolution in minimally perturbed naive non-transparent samples has recently gained increased popularity and broad application in biomedical research. Here, we describe a novel X-ray-based methodology for analysis of ß-galactosidase (lacZ) reporter-driven gene expression in an intact murine brain ex vivo by micro-CT. The method relies on detection of bromine molecules in the product of the enzymatic ß-galactosidase reaction. Enhancement of the X-ray signal is observed specifically in the regions of the murine brain where expression of the lacZ reporter gene is also detected histologically. We performed quantitative analysis of the expression levels of lacZ reporter activity by relative radiodensity estimation of the ß-galactosidase/X-gal precipitate in situ. To demonstrate the feasibility of the method, we performed expression analysis of the Tsen54-lacZ reporter gene in the murine brain in a semi-quantitative manner. Human mutations in the Tsen54 gene cause pontocerebellar hypoplasia (PCH), a group of severe neurodegenerative disorders with both mental and motor deficits. Comparing relative levels of Tsen54 gene expression, we demonstrate that the highest Tsen54 expression is observed in anatomical brain substructures important for the normal motor and memory functions in mice.

Functional loss of Ccdc151 leads to hydrocephalus in a mouse model of primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder affecting normal structure and function of motile cilia, phenotypically manifested as chronic respiratory infections, laterality defects and infertility. Autosomal recessive mutations in genes encoding for different components of the ciliary axoneme have been associated with PCD in humans and in model organisms. The CCDC151 gene encodes for a coiled-coil axonemal protein that ensures correct attachment of outer dynein arm (ODA) complexes to microtubules. A correct arrangement of dynein arm complexes is required to provide the proper mechanical force necessary for cilia beat. Loss-of-function mutations in CCDC151 in humans leads to PCD disease with respiratory distress and defective left-right body asymmetry. In mice with the Ccdc151Snbl loss-of-function mutation (Snowball mutant), left-right body asymmetry with heart defects have been observed. Here, we demonstrate that loss of Ccdc151 gene function via targeted gene deletion in mice leads to perinatal lethality and congenital hydrocephalus. Microcomputed tomography (microCT) X-ray imaging of Ccdc151-ß-galactosidase reporter expression in whole-mount brain and histological analysis show that Ccdc151 is expressed in ependymal cells lining the ventricular brain system, further confirming the role of Ccdc151 dysfunction in hydrocephalus development. Analyzing the features of hydrocephalus in the Ccdc151-knockout animals by microCT volumetric imaging, we observe continuity of the aqueduct of Sylvius, indicating the communicating nature of hydrocephalus in the Ccdc151-knockout animals. Congenital defects in left-right asymmetry and male infertility have been also observed in Ccdc151-null animals. Ccdc151 gene deletion in adult animals results in abnormal sperm counts and defective sperm motility.This article has an associated First Person interview with the joint first authors of the paper.

Insights into specificity, redundancy and new cellular functions of C/EBPa and C/EBPb transcription factors through interactome network analysis.

BACKGROUND: C/EBPa and C/EBPb are transcription factors with tissue specific expression regulating several important cellular processes. They work by recruiting protein complexes to a common DNA recognition motif and both are able to compensate each other's absence in many cell types, thus showing functional redundancy. They also play distinct roles in specific cellular pathways and their abnormal functioning gives raise to different human pathologies. METHODS: To investigate the molecular basis of C/EBPa and C/EBPb specificity and redundancy we characterized their in vivo protein-protein interaction networks by Tandem Affinity Purification (TAP) and Mass Spectrometry (MS). To unravel the functional features of C/EBPa and C/EBPb proteomes we studied the statistical enrichment of binding partners related to Gene Ontology (GO) terms and KEGG pathways. RESULTS: Our data confirmed that the C/EBPa and C/EBPb regulate biological processes like cell proliferation, apoptosis and transformation. We found that both C/EBPa and C/EBPb are involved in other cellular pathways such as RNA maturation, RNA splicing and DNA repair. Specific interactions of C/EBPa with MRE11, RUVBL1 and RUVBL2 components of DNA repair system were confirmed by co-immunoprecipitation assays. CONCLUSIONS: Our comparative analysis of the C/EBPa and C/EBPb proteomes provides an insight for understanding both their redundant and specific roles in cells indicating their involvement in new pathways. Such novel predicted functions are relevant to normal cellular processes and disease phenotypes controlled by these transcription factors. GENERAL SIGNIFICANCE: Functional characterization of C/EBPa and C/EBPb proteomes suggests they can regulate novel pathways and indicate potential molecular targets for therapeutic intervention.

Role of the amino sugar in the DNA binding of disaccharide anthracyclines: crystal structure of the complex MAR70/d(CGATCG).

Disaccharide anthracyclines analogues have been shown to exhibit different antitumour activity as compared with parents compounds doxorubicin and daunomycin. Here we report the crystal structure of the disaccharide analog MAR70 complexed with the DNA hexamer d(CGATCG). The structure has been solved at 1.54A resolution and is similar to previous crystallized anthracycline-DNA complexes with both sugar rings of the disaccharide chain lying in the DNA minor groove. Comparison with the structure of MEN10755 another disaccharide anthracycline co-crystallized with the same DNA hexamer suggests a correlation between the position of the amino sugar on the disaccharide chain and the conformation of this moiety when binding to DNA. This is discussed with respect to the influence on drug activity and on the possible interaction with other cellular targets.

ABAD directly links Abeta to mitochondrial toxicity in Alzheimer's disease.

Mitochondrial dysfunction is a hallmark of beta-amyloid (Abeta)-induced neuronal toxicity in Alzheimer's disease (AD). Here, we demonstrate that Abeta-binding alcohol dehydrogenase (ABAD) is a direct molecular link from Abeta to mitochondrial toxicity. Abeta interacts with ABAD in the mitochondria of AD patients and transgenic mice. The crystal structure of Abeta-bound ABAD shows substantial deformation of the active site that prevents nicotinamide adenine dinucleotide (NAD) binding. An ABAD peptide specifically inhibits ABAD-Abeta interaction and suppresses Abeta-induced apoptosis and free-radical generation in neurons. Transgenic mice overexpressing ABAD in an Abeta-rich environment manifest exaggerated neuronal oxidative stress and impaired memory. These data suggest that the ABAD-Abeta interaction may be a therapeutic target in AD.

The three-dimensional structures of the Mycobacterium tuberculosis dihydrodipicolinate reductase-NADH-2,6-PDC and -NADPH-2,6-PDC complexes. Structural and mutagenic analysis of relaxed nucleotide specificity.

Dihydrodipicolinate reductase (DHPR) catalyzes the reduced pyridine nucleotide-dependent reduction of the alpha,beta-unsaturated cyclic imine, dihydrodipicolinate, to generate tetrahydrodipicolinate. This enzyme catalyzes the second step in the bacterial biosynthetic pathway that generates meso-diaminopimelate, a component of bacterial cell walls, and the amino acid L-lysine. The Mycobacterium tuberculosis dapB-encoded DHPR has been cloned, expressed, purified, and crystallized in two ternary complexes with NADH or NADPH and the inhibitor 2,6-pyridinedicarboxylate (2,6-PDC). The structures have been solved using molecular replacement strategies, and the DHPR-NADH-2,6-PDC and DHPR-NADPH-2,6-PDC complexes have been refined against data to 2.3 and 2.5 A, respectively. The M. tuberculosis DHPR is a tetramer of identical subunits, with each subunit composed of two domains connected by two flexible hinge regions. The N-terminal domain binds pyridine nucleotide, while the C-terminal domain is involved in both tetramer formation and substrate/inhibitor binding. The M. tuberculosis DHPR uses NADH and NADPH with nearly equal efficiency based on V/K values. To probe the nature of this substrate specificity, we have generated two mutants, K9A and K11A, residues that are close to the 2'-phosphate of NADPH. These two mutants exhibit decreased specificity for NADPH by factors of 6- and 30-fold, respectively, but the K11A mutant exhibits 270% of WT activity using NADH. The highly conserved structure of the nucleotide fold may permit other enzyme's nucleotide specificity to be altered using similar mutagenic strategies.

The use of construct variation and diffraction data analysis in the crystallization of the TRAF domain of human tumor necrosis factor receptor associated factor 6.

Obtaining soluble proteins and diffraction-quality crystals often represents the bottleneck in macromolecular crystallography. Here, it is shown that construct variation can be an efficient strategy in expressing soluble proteins from bacteria and that clues from poor crystals may be used to improve crystal packing and to optimize crystal quality.

Distinct molecular mechanism for initiating TRAF6 signalling.

Tumour-necrosis factor (TNF) receptor-associated factor 6 (TRAF6) is the only TRAF family member that participates in signal transduction of both the TNF receptor (TNFR) superfamily and the interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) superfamily; it is important for adaptive immunity, innate immunity and bone homeostasis. Here we report crystal structures of TRAF6, alone and in complex with TRAF6-binding peptides from CD40 and TRANCE-R (also known as RANK), members of the TNFR superfamily, to gain insight into the mechanism by which TRAF6 mediates several signalling cascades. A 40 degrees difference in the directions of the bound peptides in TRAF6 and TRAF2 shows that there are marked structural differences between receptor recognition by TRAF6 and other TRAFs. The structural determinant of the petide TRAF6 interaction reveals a Pro-X-Glu-X-X-(aromatic/acidic residue) TRAF6-binding motif, which is present not only in CD40 and TRANCE-R but also in the three IRAK adapter kinases for IL-1R/TLR signalling. Cell-permeable peptides with the TRAF6-binding motif inhibit TRAF6 signalling, which indicates their potential as therapeutic modulators. Our studies identify a universal mechanism by which TRAF6 regulates several signalling cascades in adaptive immunity, innate immunity and bone homeostasis.