Probing the Solution Structure of IκB Kinase (IKK) Subunit γ and Its Interaction with Kaposi Sarcoma-associated Herpes Virus Flice-interacting Protein and IKK Subunit β by EPR Spectroscopy

Viral flice-interacting protein (vFLIP), encoded by the oncogenic Kaposi sarcoma-associated herpes virus (KSHV), constitutively activates the canonical nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) pathway. This is achieved through subversion of the IκB kinase (IKK) complex (or signalosome), which involves a physical interaction between vFLIP and the modulatory subunit IKKγ. Although this interaction has been examined both in vivo and in vitro, the mechanism by which vFLIP activates the kinase remains to be determined. Because IKKγ functions as a scaffold, recruiting both vFLIP and the IKKα/β subunits, it has been proposed that binding of vFLIP could trigger a structural rearrangement in IKKγ conducive to activation. To investigate this hypothesis we engineered a series of mutants along the length of the IKKγ molecule that could be individually modified with nitroxide spin labels. Subsequent distance measurements using electron paramagnetic resonance spectroscopy combined with molecular modeling and molecular dynamics simulations revealed that IKKγ is a parallel coiled-coil whose response to binding of vFLIP or IKKβ is localized twisting/stiffening and not large-scale rearrangements. The coiled-coil comprises N- and C-terminal regions with distinct registers accommodated by a twist: this structural motif is exploited by vFLIP, allowing it to bind and subsequently activate the NF-κB pathway. In vivo assays confirm that NF-κB activation by vFLIP only requires the N-terminal region up to the transition between the registers, which is located directly C-terminal of the vFLIP binding site.     

Forward genetic analysis of visual behavior in zebrafish

The visual system converts the distribution and wavelengths of photons entering the eye into patterns of neuronal activity, which then drive motor and endocrine behavioral responses. The gene products important for visual processing by a living and behaving vertebrate animal have not been identified in an unbiased fashion. Likewise, the genes that affect development of the nervous system to shape visual function later in life are largely unknown. Here we have set out to close this gap in our understanding by using a forward genetic approach in zebrafish. Moving stimuli evoke two innate reflexes in zebrafish larvae, the optomotor and the optokinetic response, providing two rapid and quantitative tests to assess visual function in wild-type (WT) and mutant animals. These behavioral assays were used in a high-throughput screen, encompassing over half a million fish. In almost 2,000 F2 families mutagenized with ethylnitrosourea, we discovered 53 recessive mutations in 41 genes. These new mutations have generated a broad spectrum of phenotypes, which vary in specificity and severity, but can be placed into only a handful of classes. Developmental phenotypes include complete absence or abnormal morphogenesis of photoreceptors, and deficits in ganglion cell differentiation or axon targeting. Other mutations evidently leave neuronal circuits intact, but disrupt phototransduction, light adaptation, or behavior-specific responses. Almost all of the mutants are morphologically indistinguishable from WT, and many survive to adulthood. Genetic linkage mapping and initial molecular analyses show that our approach was effective in identifying genes with functions specific to the visual system. This collection of zebrafish behavioral mutants provides a novel resource for the study of normal vision and its genetic disorders.     

Proteomic identification of human neutrophil alpha-defensins in chronic lung allograft rejection

Chronic allograft rejection remains a leading cause of morbidity and mortality in lung transplant recipients. Currently, diagnosis is based on lung biopsies or the presence of bronchiolitis obliterans syndrome (BOS). To identify a biomarker of rejection we performed a proteome survey of archived bronchoalveolar lavage fluid (BALF) acquired from lung transplant recipients between 1993 and 1996 using mass spectrometry (MS). A total of 126 BALF samples from 57 individuals were tested. Initial MS assessment revealed numerous differences in a majority of individuals who experienced BOS, but three unusually intense peaks at m/z = 3373, 3444, and 3488. These were identified as human neutrophil peptides 1-3 (HNP). Quantification by enzyme-linked immunoabsorbent assay showed an elevated HNP level (>0.3 ng/microg protein) in 89% of patients who developed BOS2-3 within 15 months, reaching as high as 6% of the total BALF protein. In control patients, 35% demonstrated a slightly elevated HNP level that declined in all who had subsequent BALF available for testing. HNP levels did not correlate with episodes of acute rejection, cytomegalovirus or fungal infection. In conclusion, elevated HNP levels are associated with the onset of BOS and can predate the clinical onset of disease up to 15 months.     

Toe-of-slope deposits of a Givetian reef-rimmed platform: provenance of calcareous density-flow deposits (Rabat-Tiflet-Zone,Morocco)

Givetian subaqueous density-flow deposits reveal the existence of a peritidal carbonate platform in sedimentary basins preserved within the Rabat-Tiflet-Zone of Morocco. The calcareous component assemblage displays a photozoan carbonate production mode of the neritic source environments. Characteristic elements of the allochthonous faunal association are colonial tabulate corals, stromatoporoids, crinoids, bryozoans and thick-shelled brachiopods. Active growing reefs and cortoid sand shoals at the platform margin as well as periplatform carbonates at the uppermost slope settings contributed bioclastic and lithoclastic lime debris to the toe-of-slope of the carbonate apron. Bipartite cobble rudstone beds are interpreted as deposits of hyperconcentrated density flows, which cannot be maintained on very low-angle slopes for as long as more dilute flows and represent short run-out distances. Beds consisting of mostly well-organized pebbly grainstones, packstones and grainstone-wackestone couplets are deposits of surge-like concentrated flows and turbidity flows.     

Sympathetic innervation of white adipose tissue and its regulation of fat cell number

White adipose tissue (WAT) is innervated by the sympathetic nervous system (SNS), and the central origins of this innervation have been demonstrated for inguinal and epididymal WAT (iWAT and eWAT, respectively) using a viral transneuronal tract tracer, the pseudorabies virus (PRV). Although the more established role of this sympathetic innervation of WAT is as a major stimulator of lipid mobilization, this innervation also inhibits WAT fat cell number (FCN); thus, local denervation of WAT leads to marked increases in WAT mass and FCN. The purpose of this study was to extend our understanding of the SNS regulation of FCN using neuroanatomical and functional analyses. Therefore, we injected PRV into retroperitoneal WAT (rWAT) to compare the SNS outflow to this pad from what already is known for iWAT and eWAT. In addition, we tested the ability of local unilateral denervation of rWAT or iWAT to promote increases in WAT mass and FCN vs. their contralateral neurally intact counterparts. Although the overall pattern of innervation was more similar than different for rWAT vs. iWAT or eWAT, its SNS outflow appeared to involve more neurons in the suprachiasmatic and solitary tract nuclei. Denervation produced significant increases in WAT mass and FCN for both iWAT and rWAT, but FCN was increased significantly more in iWAT than in rWAT. These data suggest differences in origins of the sympathetic outflow to WAT and functional differences in the WAT SNS innervation that could contribute to the differential propensity for fat cell proliferation across WAT depots in vivo.     

X-ray structures of the maltose-maltodextrin-binding protein of the thermoacidophilic bacterium Alicyclobacillus acidocaldarius provide insight into acid stability of proteins

Maltose-binding proteins act as primary receptors in bacterial transport and chemotaxis systems. We report here crystal structures of the thermoacidostable maltose-binding protein from Alicyclobacillus acidocaldarius, and explore its modes of binding to maltose and maltotriose. Further, comparison with the structures of related proteins from Escherichia coli (a mesophile), and two hyperthermophiles (Pyrococcus furiosus and Thermococcus litoralis) allows an investigation of the basis of thermo- and acidostability in this family of proteins.The thermoacidophilic protein has fewer charged residues than the other three structures, which is compensated by an increase in the number of polar residues. Although the content of acidic and basic residues is approximately equal, more basic residues are exposed on its surface whereas most acidic residues are buried in the interior. As a consequence, this protein has a highly positive surface charge. Fewer salt bridges are buried than in the other MBP structures, but the number exposed on its surface does not appear to be unusual. These features appear to be correlated with the acidostability of the A. acidocaldarius protein rather than its thermostability.An analysis of cavities within the proteins shows that the extremophile proteins are more closely packed than the mesophilic one. Proline content is slightly higher in the hyperthermophiles and thermoacidophiles than in mesophiles, and this amino acid is more common at the second position of beta-turns, properties that are also probably related to thermostability. Secondary structural content does not vary greatly in the different structures, and so is not a contributing factor.