Autosomal dominant stapes ankylosis with broad thumbs and toes,hyperopia, and skeletal anomalies is caused by heterozygous nonsense and frameshift mutations in NOG,the gene encoding noggin

Although fixation of the stapes is usually progressive and secondary to otosclerosis, it may present congenitally, with other skeletal manifestations, as an autosomal dominant syndrome-such as proximal symphalangism (SYM1) or multiple-synostoses syndrome (SYNS1), both of which are caused by mutations in NOG, the gene encoding noggin. We describe a family that was ascertained to have nonsyndromic otosclerosis but was subsequently found to have a congenital stapes ankylosis syndrome that included hyperopia, a hemicylindrical nose, broad thumbs and great toes, and other minor skeletal anomalies but lacked symphalangism. A heterozygous nonsense NOG mutation-c.328C-->T (Q110X), predicted to truncate the latter half of the protein-was identified, and a heterozygous insertion in NOG-c.252-253insC, in which the frameshift is predicted to result in 96 novel amino acids before premature truncation-was identified in a previously described second family with a similar phenotype. In contrast to most NOG mutations that have been reported in kindreds with SYM1 and SYNS1, the mutations observed in these families with stapes ankylosis without symphalangism are predicted to disrupt the cysteine-rich C-terminal domain. These clinical and molecular findings suggest that (1) a broader range of conductive hearing-loss phenotypes are associated with NOG mutations than had previously been recognized, (2) patients with sporadic or familial nonsyndromic otosclerosis should be evaluated for mild features of this syndrome, and (3) NOG alterations should be considered in conductive hearing loss with subtle clinical and skeletal features, even in the absence of symphalangism.     

Mandibuloacral dysplasia is caused by a mutation in LMNA-encoding lamin A/C

Mandibuloacral dysplasia (MAD) is a rare autosomal recessive disorder, characterized by postnatal growth retardation, craniofacial anomalies, skeletal malformations, and mottled cutaneous pigmentation. The LMNA gene encoding two nuclear envelope proteins (lamins A and C [lamin A/C]) maps to chromosome 1q21 and has been associated with five distinct pathologies, including Dunnigan-type familial partial lipodystrophy, a condition that is characterized by subcutaneous fat loss and is invariably associated with insulin resistance and diabetes. Since patients with MAD frequently have partial lipodystrophy and insulin resistance, we hypothesized that the disease may be caused by mutations in the LMNA gene. We analyzed five consanguineous Italian families and demonstrated linkage of MAD to chromosome 1q21, by use of homozygosity mapping. We then sequenced the LMNA gene and identified a homozygous missense mutation (R527H) that was shared by all affected patients. Patient skin fibroblasts showed nuclei that presented abnormal lamin A/C distribution and a dysmorphic envelope, thus demonstrating the pathogenic effect of the R527H LMNA mutation.     

Formation of a copper specific binding site in non-native states of beta-2-microglobulin

A debilitating complication of long-term hemodialysis is the deposition of beta-2-microglobulin (beta2m) as amyloid plaques in the joint space. We have recently shown that Cu(2+) can be a contributing, if not causal, factor at concentrations encountered during dialysis therapy. The basis for this effect is destabilization and incorporation of beta2m into amyloid fibers upon binding of Cu(2+). In this work, we demonstrate that while beta2m binds Cu(2+) specifically in the native state, it is binding of Cu(2+) by non-native states of beta2m which is responsible for destabilization. Mutagenesis of potential coordinating groups for Cu(2+) shows that native state binding of Cu(2+) is mediated by residues and structures that are different than those which bind in non-native states. An increased affinity for copper by non-native states compared to that of the native state gives rise to overall destabilization. Using mass spectrometry, NMR, and fluorescence techniques, we show that native state binding is localized to H31 and W60 and is highly specific for Cu(2+) over Zn(2+) and Ni(2+). Binding of Cu(2+) in non-native states of beta2m is mediated by residues H13, H51, and H84, but not H31. Although denatured beta2m has characteristics of a globally unfolded state, it nevertheless demonstrates the following strong specificity of binding: Cu(2+) > Zn(2+) > Ni(2+). This requires the existence of a well-defined structure in the unfolded state of this protein. As Cu(2+) effects are reported in many other amyloidoses, e.g., PrP, alpha-synuclein, and Abeta, our results may be extended to the emerging field of divalent ion-associated amyloidosis.     

Multiparasitism of Choristoneura fumiferana by the ichneumonid Tranosema rostrale and the tachinid Actia interrupta: occurrence in the field and outcome of competition under laboratory conditions

Tranosema rostrale (Brishke) (Hymenoptera, Ichneumonidae) and Actia interrupta Curran (Hymenoptera: Tachinidae) are the two endoparasitoids most frequently encountered in low-density populations of the spruce budworm, Choristoneura fumiferana (Clemens) (Lepidoptera, Tortricidae), in the Quebec City region. Monitoring of attack rates of implanted C. fumiferana larvae at two different study sites suggested the possible existence of competition between the two parasitoids, with A. interrupta seemingly displacing T. rostrale. Here, we show that multiparasitism involving these two species does occur in the field, but at a frequency too low to explain the seasonal pattern of decline in apparent parasitism by T. rostrale that accompanies the rise of A. interrupta attack rates. We also provide preliminary evidence, from laboratory experiments, that A. interrupta has a competitive advantage over T. rostrale and that the success of parasitism by A. interrupta may be enhanced by prior parasitism by T. rostrale under certain conditions, possibly due to the presence of the latter species' polydnavirus. In addition, we describe a PCR-based method that we developed to help detect the presence of T. rostrale eggs which often escape detection by simple visual examination of the dissected host larvae; DNA sequences specific to the polydnavirus injected by the female wasp at the time of oviposition can be readily amplified from whole host larvae.     

Crystal structure of Escherichia coli glucose-1-phosphate thymidylyltransferase (RffH) complexed with dTTP and Mg2+

The enzyme glucose-1-phosphate thymidylyltransferase (RffH), the product of the rffh gene, catalyzes one of the steps in the synthesis of enterobacterial common antigen (ECA), a cell surface glycolipid found in Gram-negative enteric bacteria. In Escherichia coli two gene products, RffH and RmlA, catalyze the same enzymatic reaction and are homologous in sequence; however, they are part of different operons and function in different pathways. We report the crystal structure of RffH bound to deoxythymidine triphosphate (dTTP), the phosphate donor, and Mg(2+), refined at 2.6 A to an R-factor of 22.3% (R(free) = 28.4%). The crystal structure of RffH shows a tetrameric enzyme best described as a dimer of dimers. Each monomer has an overall alpha/beta fold and consists of two domains, a larger nucleotide binding domain (residues 1-115, 222-291) and a smaller sugar-binding domain (116-221), with the active site located at the domain interface. The Mg(2+) ion is coordinated by two conserved aspartates and the alpha-phosphate of deoxythymidine triphosphate. Its location corresponds well to that in a structurally similar domain of N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU). Analysis of the RffH, RmlA, and GlmU complexes with substrates and products provides an explanation for their different affinities for Mg(2+) and leads to a proposal for the dynamics along the reaction pathway.     

Novel interaction between the M4 muscarinic acetylcholine receptor and elongation factor 1A2

The activation of the muscarinic acetylcholine receptor (mAChR) family, consisting of five subtypes (M1-M5), produces a variety of physiological effects throughout the central nervous system. However, the role of each individual subtype remains poorly understood. To further elucidate signal transduction pathways for specific subtypes, we used the most divergent portion of the subtypes, the intracellular third (i3) loop, as bait to identify interacting proteins. Using a brain pull-down assay, we identify elongation factor 1A2 (eEF1A2) as a specific binding partner to the i3 loop of M4, and not to M1 or M2. In addition, we demonstrate a direct interaction between these proteins. In the rat striatum, the M4 mAChR colocalizes with eEF1A2 in the soma and neuropil. In PC12 cells, endogenous eEF1A2 co-immunoprecipitates with the endogenous M4 mAChR, but not with the endogenous M1 mAChR. In our in vitro model, M4 dramatically accelerates nucleotide exchange of eEF1A2, a GTP-binding protein. This indicates the M4 mAChR is a guanine exchange factor for eEF1A2. eEF1A2 is an essential GTP-binding protein for protein synthesis. Thus, our data suggest a novel role for M4 in the regulation of protein synthesis through its interaction with eEF1A2.     

Kinetic mechanisms of IkappaB-related kinases (IKK) inducible IKK and TBK-1 differ from IKK-1/IKK-2 heterodimer

Nuclear factor-kappaB activation depends on phosphorylation and degradation of its inhibitor protein, IkappaB. The phosphorylation of IkappaBalpha on Ser(32) and Ser(36) is initiated by an IkappaB kinase (IKK) complex that includes a catalytic heterodimer composed of IkappaB kinase 1 (IKK-1) and IkappaB kinase 2 (IKK-2) as well as a regulatory adaptor subunit, NF-kappaB essential modulator. Recently, two related IkappaB kinases, TBK-1 and IKK-i, have been described. TBK-1 and IKK-i show sequence and structural homology to IKK-1 and IKK-2. TBK-1 and IKK-i phosphorylate Ser(36) of IkappaBalpha. We describe the kinetic mechanisms in terms of substrate and product inhibition of the recombinant human (rh) proteins, rhTBK-1, rhIKK-I, and rhIKK-1/rhIKK-2 heterodimers. The results indicate that although each of these enzymes exhibits a random sequential kinetic mechanism, the effect of the binding of one substrate on the affinity of the other substrate is significantly different. ATP has no effect on the binding of an IkappaBalpha peptide for the rhIKK-1/rhIKK-2 heterodimer (alpha = 0.99), whereas the binding of ATP decreased the affinity of the IkappaBalpha peptide for both rhTBK-1 (alpha = 10.16) and rhIKK-i (alpha = 62.28). Furthermore, the dissociation constants of ATP for rhTBK-1 and rhIKK-i are between the expected values for kinases, whereas the dissociation constants of the IkappaBalpha peptide for each IKK isoforms is unique with rhTBK-1 being the highest (K(IkappaBalpha) = 69.87 microm), followed by rhIKK-i (K(IkappaBalpha) = 5.47 microm) and rhIKK-1/rhIKK-2 heterodimers (K(IkappaBalpha) = 0.12 microm). Thus this family of IkappaB kinases has very unique kinetic properties.     

Apolipoprotein A-I alpha -helices 7 and 8 modulate high density lipoprotein subclass distribution.

Mice have a monodisperse high density lipoprotein (HDL) profile, whereas humans have two major subfractions designated HDL(2) and HDL(3). Human apoA-I transgenic mice exhibit a human-like HDL profile, indicating that the amino acid sequence of apoA-I is a determinant of the HDL profile. Comparison of the primary sequence of mouse and human apoA-I and the previously designated "hinge" domain of apoA-I led us to hypothesize that alpha-helices 7 and 8 (7/8) are determinants of HDL subclass distribution. The following proteins were expressed in Escherichia coli: human apoA-I, T7-hAI; mouse apoA-I, T7-mAI; chimeric human apoA-I containing murine helices 7/8 in place of human helices 7/8, T7-hAI(m7/8); and the reciprocal chimera, T7-mAI(h7/8). The recombinant proteins were examined for their association with human plasma HDL subclasses. The results demonstrated that T7-hAI bound HDL(2) and HDL(3) equally well, whereas T7-mAI bound to HDL(2) preferentially. T7-hAI(m7/8) behaved like T7-mAI, and T7-mAI(h7/8) behaved like T7-hAI. Thus, alpha-helices 7/8 are strong contributors to the pattern of HDL subclass association. Self-association, alpha-helicity, cholesterol efflux, and lecithin-cholesterol acyltransferase activity of the recombinant proteins were also assessed. Human apoA-I self-associates more and activates human lecithin-cholesterol acyltransferase better than mouse apoA-I. These differential characteristics of human and mouse apoA-I are not dependent on helices 7/8.     

Reduction of atherosclerosis by the peroxisome proliferator-activated receptor alpha agonist fenofibrate in mice

Several clinical and angiographic intervention trials have shown that fibrate treatment leads to a reduction of the coronary events associated to atherosclerosis. Fibrates are ligands for peroxisome proliferator-activated receptor alpha (PPARalpha) that modulate risk factors related to atherosclerosis by acting at both systemic and vascular levels. Here, we investigated the effect of treatment with the PPARalpha agonist fenofibrate (FF) on the development of atherosclerotic lesions in apolipoprotein (apo) E-deficient mice and human apoA-I transgenic apoE-deficient (hapoA-I Tg x apoE-deficient) mice fed a Western diet. In apoE-deficient mice, plasma lipid levels were increased by FF treatment with no alteration in the cholesterol distribution profile. FF treatment did not reduce atherosclerotic lesion surface area in the aortic sinus of 5-month-old apoE-deficient mice. By contrast, FF treatment decreased total cholesterol and esterified cholesterol contents in descending aortas of these mice, an effect that was more pronounced in older mice exhibiting more advanced lesions. Furthermore, FF treatment reduced MCP-1 mRNA levels in the descending aortas of apoE-deficient mice, whereas ABCA-1 expression levels were maintained despite a significant reduction of aortic cholesterol content. In apoE-deficient mice expressing a human apoA-I transgene, FF increased human apoA-I plasma and hepatic mRNA levels without affecting plasma lipid levels. This increase in human apoA-I expression was accompanied by a significant reduction in the lesion surface area in the aortic sinus. These data indicate that the PPARalpha agonist fenofibrate reduces atherosclerosis in these animal models of atherosclerosis.