Mice lacking the UbCKmit isoform of creatine kinase reveal slower spatial learning acquisition,diminished exploration and habituation,and reduced acoustic startle reflex responses

Brain-type creatine kinases B-CK (cytosolic) and UbCKmit (mitochondrial) are considered important for the maintenance and distribution of cellular energy in the central nervous system. Previously, we have demonstrated an abnormal behavioral phenotype in mice lacking the B-CK creatine kinase isoform, regarding exploration, habituation, seizure susceptibility and spatial learning. The phenotype in these mice was associated with histological adaptations in the hippocampal mossy fiber field size. Here, mice lacking the ubiquitous mitochondrial creatine kinase isoform (UbCKmit-/- mice) showed, when subjected to a similar battery of behavioral tasks, diminished open field habituation and slower spatial learning acquisition in the Morris water maze task, but normal sensory or motor functions. A reduced acoustic startle response, higher threshold, and lack of prepulse inhibition were observed in UbCKmit-/- mice, suggesting that the unconditioned reflexive responsiveness is not optimal. Our findings suggest a role for mitochondrial CK-mediated high-energy phosphoryl transfer in synaptic signalling in the acoustic signal response network and hippocampal-dependent learning circuitry of brain. Finally, we demonstrate that UbCKmit has a widespread occurrence in the cell soma of neuronal nuclei along the rostro-caudal axis of the brain, i.e. cortex, midbrain, hindbrain, cerebellum and brainstem, similar to the occurrence of B-CK. This may explain the similarity of phenotypes in mice lacking B-CK or UbCKmit. We predict that the remaining functional intactness of the cytosolic B-CK reaction and perhaps the compensatory role of other phosphoryl transfer systems are sufficient to sustain the energy requirements for basic sensory, motor and physiological activities in UbCKmit-/- mice.     

Photoisomerization and photoionization of the photoactive yellow protein chromophore in solution

Dispersed pump-dump-probe spectroscopy has the ability to characterize and identify the underlying ultrafast dynamical processes in complicated chemical and biological systems. This technique builds on traditional pump-probe techniques by exploring both ground- and excited-state dynamics and characterizing the connectivity between constituent transient states. We have used the dispersed pump-dump-probe technique to investigate the ground-state dynamics and competing excited-state processes in the excitation-induced ultrafast dynamics of thiomethyl p-coumaric acid, a model chromophore for the photoreceptor photoactive yellow protein. Our results demonstrate the parallel formation of two relaxation pathways (with multiple transient states) that jointly lead to two different types of photochemistry: cis-trans isomerization and detachment of a hydrated electron. The relative transition rates and quantum yields of both pathways have been determined. We find that the relaxation of the photoexcited chromophores involves multiple, transient ground-state intermediates and the chromophore in solution does not generate persistent photoisomerized products, but instead undergoes photoionization resulting in the generation of detached electrons and radicals. These results are of great value in interpreting the more complex dynamical changes in the optical properties of the photoactive yellow protein.     

Nonmuscle myosin heavy-chain gene MYH14 is expressed in cochlea and mutated in patients affected by autosomal dominant hearing impairment (DFNA4)

Myosins have been implicated in various motile processes, including organelle translocation, ion-channel gating, and cytoskeleton reorganization. Different members of the myosin superfamily are responsible for syndromic and nonsyndromic hearing impairment in both humans and mice. MYH14 encodes one of the heavy chains of the class II nonmuscle myosins, and it is localized within the autosomal dominant hearing impairment (DFNA4) critical region. After demonstrating that MYH14 is highly expressed in mouse cochlea, we performed a mutational screening in a large series of 300 hearing-impaired patients from Italy, Spain, and Belgium and in a German kindred linked to DFNA4. This study allowed us to identify a nonsense and two missense mutations in large pedigrees, linked to DFNA4, as well as a de novo allele in a sporadic case. Absence of these mutations in healthy individuals was tested in 200 control individuals. These findings clearly demonstrate the role of MYH14 in causing autosomal dominant hearing loss and further confirm the crucial role of the myosin superfamily in auditive functions.     

Rap2B-dependent stimulation of phospholipase C-epsilon by epidermal growth factor receptor mediated by c-Src phosphorylation of RasGRP3

Receptor tyrosine kinase regulation of phospholipase C-epsilon (PLC-epsilon), which is under the control of Ras-like and Rho GTPases, was studied with HEK-293 cells endogenously expressing PLC-coupled epidermal growth factor (EGF) receptors. PLC and Ca(2+) signaling by the EGF receptor, which activated both PLC-gamma1 and PLC-epsilon, was specifically suppressed by inactivation of Ras-related GTPases with clostridial toxins and expression of dominant-negative Rap2B. EGF induced rapid and sustained GTP loading of Rap2B, binding of Rap2B to PLC-epsilon, and Rap2B-dependent translocation of PLC-epsilon to the plasma membrane. GTP loading of Rap2B by EGF was inhibited by chelation of intracellular Ca(2+) and expression of lipase-inactive PLC-gamma1 but not of PLC-epsilon. Expression of RasGRP3, a Ca(2+)/diacylglycerol-regulated guanine nucleotide exchange factor for Ras-like GTPases, but not expression of various other exchange factors enhanced GTP loading of Rap2B and PLC/Ca(2+) signaling by the EGF receptor. EGF induced tyrosine phosphorylation of RasGRP3, but not RasGRP1, apparently caused by c-Src; inhibition of c-Src interfered with EGF-induced Rap2B activation and PLC stimulation. Collectively, these data suggest that the EGF receptor triggers activation of Rap2B via PLC-gamma1 activation and tyrosine phosphorylation of RasGRP3 by c-Src, finally resulting in stimulation of PLC-epsilon.     

Oligomeric assembly of native-like precursors precedes amyloid formation by beta-2 microglobulin

The deposition of beta-2-microglobulin (beta2m) as amyloid fibers results in debilitating complications for renal failure patients who are treated by hemodialysis. In vitro, wild-type beta2m can be converted to amyloid under physiological conditions by exposure to biomedically relevant concentrations of Cu(2+). In this work, we have made comparative measurements of the structural and oligomeric changes in beta2m at time points preceding fibrillogenesis. Our results show Cu(2+) mediates the formation of a monomeric, activated state followed by the formation of a discrete dimeric intermediate. The dimeric intermediates then assemble into tetra- and hexameric forms which display little additional oligomerization on the time scales of their own formation (<1 h). Amyloid fiber formation progresses from these intermediate states but on much longer time scales (>1 week). Although Cu(2+) is necessary for the generation and stabilization of these intermediates, it is not required for the stability of mature amyloid fibers. This suggests that Cu(2+) acts as an initiating factor of amyloidosis by inducing oligomer formation. (1)H NMR and near-UV circular dichroism are used to establish that oligomeric intermediates are native-like in structure. The native-like structure and discrete oligomeric size of beta2m amyloid intermediates suggest that this protein forms fibrils by structural domain swapping.     

Molecular dynamics simulations of the lipid bilayer edge

Phospholipid bilayers have been intensively studied by molecular dynamics (MD) simulation in recent years. The properties of bilayer edges are important in determining the structure and stability of pores formed in vesicles and biomembranes. In this work, we use molecular dynamics simulation to investigate the structure, dynamics, and line tension of the edges of bilayer ribbons composed of pure dimyristoylphosphatidylcholine (DMPC) or palmitoyl-oleoylphosphatidylethanolamine (POPE). As expected, we observe a significant reorganization of lipids at and near the edges. The treatment of electrostatic effects is shown to have a qualitative impact on the structure and stability of the edge, and significant differences are observed in the dynamics and structure of edges formed by DMPC and palmitoyl-oleoylphosphatidylethanolamine. From the pressure anisotropy in the simulation box, we calculate a line tension of approximately 10-30 pN for the DMPC edge, in qualitative agreement with experimental estimates for similar lipids.     

Position of single amino acid substitutions in the collagen triple helix determines their effect on structure of collagen fibrils

Collagen II fibrils are a critical structural component of the extracellular matrix of cartilage providing the tissue with its unique biomechanical properties. The self-assembly of collagen molecules into fibrils is a spontaneous process that depends on site-specific binding between specific domains belonging to interacting molecules. These interactions can be altered by mutations in the COL2A1 gene found in patients with a variety of heritable cartilage disorders known as chondrodysplasias. Employing recombinant procollagen II, we studied the effects of R75C or R789C mutations on fibril formation. We determined that both R75C and R789C mutants were incorporated into collagen assemblies. The effects of the R75C and R789C substitutions on fibril formation differed significantly. The R75C substitution located in the thermolabile region of collagen II had no major effect on the fibril formation process or the morphology of fibrils. In contrast, the R789C substitution located in the thermostable region of collagen II caused profound changes in the morphology of collagen assemblies. These results provide a basis for identifying pathways leading from single amino acid substitutions in collagen II to changes in the structure of individual fibrils and in the organization of collagenous matrices.     

Mechanism of the dihydroorotase reaction

Dihydroorotase (DHO) is a zinc metalloenzyme that functions in the pathway for the biosynthesis of pyrimidine nucleotides by catalyzing the reversible interconversion of carbamoyl aspartate and dihydroorotate. A chemical mechanism was proposed on the basis of an analysis of the effects of pH, metal substitution, solvent isotope effects, mutant proteins, and alternative substrates on the enzyme-catalyzed reaction. The pH-rate profiles for the hydrolysis of dihydroorotate or thiodihydroorotate demonstrated that a single group from the enzyme must be unprotonated for maximal catalytic activity. Conversely, the pH-rate profiles for the condensation of carbamoyl aspartate to dihydroorotate showed that a single group from the enzyme must be protonated for maximal catalytic activity. The native zinc ions within the active site of DHO were substituted with cobalt or cadmium by reconstitution of the apoenzyme with divalent cations in the presence of bicarbonate. The ionizations observed in the pH-rate profiles were dependent on the specific metal ion bound to the active site. Mutation of the residue (Asp-250) that hydrogen bonds to the bridging hydroxide (or water) resulted in the loss of catalytic activity. These results are consistent with the formation of a hydroxide bridge between the two divalent cations that functions as the nucleophile during the hydrolysis of dihydroorotate. In addition, Asp-250 is postulated to shuttle the proton from the bridging hydroxide to the leaving group amide during hydrolysis of dihydroorotate. The X-ray crystal structure of DHO showed that the exocyclic alpha-carboxylate of dihydroorotate is bound to the protein via electrostatic interactions with Arg-20, Asn-44, and His-254. Mutation of these residues resulted in the loss of catalytic activity, indicating that these residues are critical for substrate recognition. The thio analogue of dihydroorotate was found to be a good substrate of the enzyme. A comprehensive chemical mechanism for DHO was proposed on the basis of the experimental findings in this study and the X-ray crystal structure.     

Inhibition of peptide bond formation by pleuromutilins: the structure of the 50S ribosomal subunit from Deinococcus radiodurans in complex with tiamulin

Tiamulin, a prominent member of the pleuromutilin class of antibiotics, is a potent inhibitor of protein synthesis in bacteria. Up to now the effect of pleuromutilins on the ribosome has not been determined on a molecular level. The 3.5 A structure of the 50S ribosomal subunit from Deinococcus radiodurans in complex with tiamulin provides for the first time a detailed picture of its interactions with the 23S rRNA, thus explaining the molecular mechanism of the antimicrobial activity of the pleuromutilin class of antibiotics. Our results show that tiamulin is located within the peptidyl transferase center (PTC) of the 50S ribosomal subunit with its tricyclic mutilin core positioned in a tight pocket at the A-tRNA binding site. Also, the extension, which protrudes from its mutilin core, partially overlaps with the P-tRNA binding site. Thereby, tiamulin directly inhibits peptide bond formation. Comparison of the tiamulin binding site with other PTC targeting drugs, like chloramphenicol, clindamycin and streptogramins, may facilitate the design of modified or hybridized drugs that extend the applicability of this class of antibiotics.     

Differential Function of Lip Residues in the Mechanism and Biology of an Anthrax Hemophore

To replicate in mammalian hosts, bacterial pathogens must acquire iron. The majority of iron is coordinated to the protoporphyrin ring of heme, which is further bound to hemoglobin. Pathogenic bacteria utilize secreted hemophores to acquire heme from heme sources such as hemoglobin. Bacillus anthracis, the causative agent of anthrax disease, secretes two hemophores, IsdX1 and IsdX2, to acquire heme from host hemoglobin and enhance bacterial replication in iron-starved environments. Both proteins contain NEAr-iron Transporter (NEAT) domains, a conserved protein module that functions in heme acquisition in Gram-positive pathogens. Here, we report the structure of IsdX1, the first of a Gram-positive hemophore, with and without bound heme. Overall, IsdX1 forms an immunoglobin-like fold that contains, similar to other NEAT proteins, a 3₁₀-helix near the heme-binding site. Because the mechanistic function of this helix in NEAT proteins is not yet defined, we focused on the contribution of this region to hemophore and NEAT protein activity, both biochemically and biologically in cultured cells. Site-directed mutagenesis of amino acids in and adjacent to the helix identified residues important for heme and hemoglobin association, with some mutations affecting both properties and other mutations affecting only heme stabilization. IsdX1 with mutations that reduced the ability to associate with hemoglobin and bind heme failed to restore the growth of a hemophore-deficient strain of B. anthracis on hemoglobin as the sole iron source. These data indicate that not only is the 3₁₀-helix important for NEAT protein biology, but also that the processes of hemoglobin and heme binding can be both separate as well as coupled, the latter function being necessary for maximal heme-scavenging activity. These studies enhance our understanding of NEAT domain and hemophore function and set the stage for structure-based inhibitor design to block NEAT domain interaction with upstream ligands.