Using Sequence Similarity Networks for Visualization of Relationships Across Diverse Protein Superfamilies

The dramatic increase in heterogeneous types of biological data—in particular, the abundance of new protein sequences—requires fast and user-friendly methods for organizing this information in a way that enables functional inference. The most widely used strategy to link sequence or structure to function, homology-based function prediction, relies on the fundamental assumption that sequence or structural similarity implies functional similarity. New tools that extend this approach are still urgently needed to associate sequence data with biological information in ways that accommodate the real complexity of the problem, while being accessible to experimental as well as computational biologists. To address this, we have examined the application of sequence similarity networks for visualizing functional trends across protein superfamilies from the context of sequence similarity. Using three large groups of homologous proteins of varying types of structural and functional diversity—GPCRs and kinases from humans, and the crotonase superfamily of enzymes—we show that overlaying networks with orthogonal information is a powerful approach for observing functional themes and revealing outliers. In comparison to other primary methods, networks provide both a good representation of group-wise sequence similarity relationships and a strong visual and quantitative correlation with phylogenetic trees, while enabling analysis and visualization of much larger sets of sequences than trees or multiple sequence alignments can easily accommodate. We also define important limitations and caveats in the application of these networks. As a broadly accessible and effective tool for the exploration of protein superfamilies, sequence similarity networks show great potential for generating testable hypotheses about protein structure-function relationships.     

Membrane stretch slows the concerted step prior to opening in a Kv channel

In the simplest model of channel mechanosensitivity, expanded states are favored by stretch. We showed previously that stretch accelerates voltage-dependent activation and slow inactivation in a Kv channel, but whether these transitions involve expansions is unknown. Thus, while voltage-gated channels are mechanosensitive, it is not clear whether the simplest model applies. For Kv pore opening steps, however, there is excellent evidence for concerted expansion motions. To ask how these motions respond to stretch, therefore, we have used a Kv1 mutant, Shaker ILT, in which the step immediately prior to opening is rate limiting for voltage-dependent current. Macroscopic currents were measured in oocyte patches before, during, and after stretch. Invariably, and directly counter to prediction for expansion-derived free energy, ILT current activation (which is limited by the concerted step prior to pore opening) slowed with stretch and the g(V) curve reversibly right shifted. In WTIR (wild type, inactivation removed), the g(V) (which reflects independent voltage sensor motions) is left shifted. Stretch-induced slowing of ILT activation was fully accounted for by a decreased basic forward rate, with no change of gating charge. We suggest that for the highly cooperative motions of ILT activation, stretch-induced disordering of the lipid channel interface may yield an entropy increase that dominates over any stretch facilitation of expanded states. Since tail current tau(V) reports on the opposite (closing) motions, ILT and WTIR tau(V)(tail) were determined, but the stretch responses were too complex to shed much light. Shaw is the Kv3 whose voltage sensor, introduced into Shaker, forms the chimera that ILT mimics. Since Shaw2 F335A activation was reportedly a first-order concerted transition, we thought its activation might, like ILT's, slow with stretch. However, Shaw2 F335A activation proved to be sigmoid shaped, so its rate-limiting transition was not a concerted pore-opening transition. Moreover, stretch, via an unidentified non-rate-limiting transition, augmented steady-state current in Shaw2 F335A. Since putative area expansion and compaction during ILT pore opening and closing were not the energetically consequential determinants of stretch modulation, models incorporating fine details of bilayer structural forces will probably be needed to explain how, for Kv channels, bilayer stretch slows some transitions while accelerating others.     

Tunicamycin,puromycin and brefeldin A influence the subcellular distribution of neuropeptides in hypothalamic magnocellular neurones of rat

Summary Magnocellular neurones in the supraoptic nuclei of normal Long Evans and homozygous Brattleboro rats were examined electron-microscopically after intracisternal injections of tunicamycin, puromycin, or brefeldin A. Moderate (50 g) or high (200 g) doses of tunicamycin caused the formation of electron-dense filamentous accretions in the endoplasmic reticulum (ER) cisterns of vasopressin neurones, but only the high dose of tunicamycin also caused accretions to form in the ER of some oxytocin neurones. Immunogold labelling of ultrathin sections from tunicamycin-treated rats revealed that, in about 5% of vasopressin neurones, the accretions could be immunogold-labelled for vasopressin and its associated neurophysin. However, in the majority of vasopressin neurones, the sections required trypsinisation before immunolabelling of the accretions could be detected. Small accretions in the ER of oxytocin neurones did not label for oxytocin or its neurophysin without prior trypsinisation, whereas larger accretions in other oxytocin cells could be labelled without prior trypsin treatment. Administration of puromycin resulted in the formation of small ER accretions in both vasopressin and oxytocin neurones. These accretions were immunolabelled with antisera, respectively, to vasopressin and oxytocin, but neurophysin-immunoreactivity was in most cases absent and was not revealed by treatment with trypsin, suggesting that neurophysin-immunoreactive epitopes were absent from truncated peptides forming the accretions. Brefeldin A caused dilatation of ER cisterns and disruption of the Golgi apparatus in both oxytocin and vasopressin neurones, but did not cause accretions to form in the ER.     

Gene expression profiles and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells

Microarray analysis is a powerful tool to identify the biological effects of drugs or chemicals on cellular gene expression. In this study, we compare the relationships between traditional measures of genetic toxicology and mutagen-induced alterations in gene expression profiles. TK6 cells were incubated with 0.01, 0.1, or 1.0 microM +/-anti-benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE) for 4 h and then cultured for an additional 20 h. Aliquots of the exposed cells were removed at 4 and 24 h in order to quantify DNA adduct levels by 32P post-labeling and measure cell viability by cloning efficiency and flow cytometry. Gene expression profiles were developed by extracting total RNA from the control and exposed cells at 4 and 24 h, labeling with Cy3 or Cy5 and hybridizing to a human 350 gene array. Mutant frequencies in the Thymidine Kinase and Hypoxanthine Phosphoribosyl Transferase genes were also determined. The 10alpha-(deoxyguanosin-N(2)-yl)-7alpha,8beta,9beta-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene (dG-N(2)-BPDE) adduct increased as a function of dose and was the only adduct identified. A dose-related decrease in cell viability was evident at 24 h, but not at 4 h. Cell death occurred by apoptosis. At 4 h, analysis of the gene expression profiles revealed that Glutathione Peroxidase and Gadd45 were consistently upregulated (greater than 1.5-fold and significantly (P < 0.001) greater than the control in two experiments) in response to 1.0 microM BPDE exposure. Fifteen genes were consistently down-regulated (less than 0.67-fold and significantly (P < 0.001) lower than the control in two experiments) at 4 h in cultures exposed to 1.0 microM BPDE. Genes with altered expression at 4 h included genes important in the progression of the cell-cycle and those that inhibit apoptosis. At 24 h post-exposure, 16 genes, involved in cell-cycle control, detoxification, and apoptosis were consistently upregulated; 10 genes were repressed in cultures exposed to the high dose of BPDE. Real-time quantitative PCR confirmed the differential expression of selected genes. These data suggest that changes in gene expression will help to identify effects of drugs and chemicals on molecular pathways in cells, and will provide useful information about the molecular responses associated with DNA damage. Of the endpoints evaluated, DNA adduct formation was the most sensitive indicator of DNA damage. DNA adduct formation was clearly evident at low doses, but the number of genes with significantly altered expression (P < 0.001) was minimal. Alterations in gene expression were more robust at doses associated with cellular toxicity and induction of mutations.     

Inactivated enzymes as probes of the structure of arabinoxylans as observed by atomic force microscopy

The complex structures of water-soluble wheat arabinoxylans have been mapped along individual molecules, and within populations, using the visualisation of the binding of inactivated enzymes by atomic force microscopy (AFM). It was demonstrated that site-directed mutagenesis (SDM) can be used to produce inactive enzymes as structural probes. For the SDM mutants AFM has been used to compare the binding of different xylanases to arabinoxylans. Xylanase mutant E386A, derived from the Xyn11A enzyme (Neocallimastrix patriciarium), was shown to bind randomly along arabinoxylan molecules. The xylanase binding was also monitored following Aspergillus niger arabinofuranosidase pre-treatment of samples. It was demonstrated that removal of arabinose side chains significantly altered the binding pattern of the inactivated enzyme. Xylanase mutant E246A, derived from the Xyn10A enzyme (Cellvibrio japonicus), was found to show deviations from random binding to the arabinoxylan chains. It is believed that this is due to the effect of a small residual catalytic activity of the enzyme that alters the binding pattern of the probe. Control procedures were developed and assessed to establish that the interactions between the modified xylanases and the arabinoxylans were specific interactions. The experimental data demonstrates the potential for using inactivated enzymes and AFM to probe the structural heterogeneity of individual polysaccharide molecules.     

Frequency-dependent distortion of meridional intensity changes during sinusoidal length oscillations of activated skeletal muscle

Bundles of intact, tetanized skeletal muscle fibers from Rana temporaria were subjected to sinusoidal length oscillations in the frequency domain 100 Hz to 3 kHz while measuring force and sarcomere length. Simultaneously, intensity of the third-order x-ray reflection of the axial myosin unit cell (I(M3)) was measured using synchrotron radiation. At oscillation frequencies <1 kHz, I(M3) was distorted during the shortening phase of the sinusoid (i.e., where bundle length was less than rest length). Otherwise, during the stretch phase of oscillations at all frequencies, during the shortening phase of oscillations above 1 kHz, and for bundles in the rigor state, I(M3) was approximately sinusoidal in form. Mean I(M3) during oscillations was reduced by 20% compared to the isometric value, suggesting a possible change in S1 disposition during oscillations. However, the amplitude of length change required to produce distortion (estimated from the phase angle at which distortion was first evident) corresponded to that of a step release sufficient to reach the maximum I(M3), indicating a mean S1 disposition during oscillations close to that during an isometric tetanus. The mechanical properties of the bundle during oscillations were also consistent with an unaltered S1 disposition during oscillations.     

Impaired functional activity of alveolar macrophages from GM-CSF-deficient mice

We hypothesized that pulmonary granulocyte-macrophage colony-stimulating factor (GM-CSF) is critically involved in determining the functional capabilities of alveolar macrophages (AM) for host defense. To test this hypothesis, cells were collected by lung lavage from GM-CSF mutant mice [GM(-/-)] and C57BL/6 wild-type mice. GM(-/-) mice yielded almost 4-fold more AM than wild-type mice. The percentage of cells positive for the beta(2)-integrins CD11a and CD11c was reduced significantly in GM(-/-) AM compared with wild-type cells, whereas expression of CD11b was similar in the two groups. The phagocytic activity of GM(-/-) AM for FITC-labeled microspheres was impaired significantly compared with that of wild-type AM both in vitro and in vivo (after intratracheal inoculation with FITC-labeled beads). Stimulated secretion of tumor necrosis factor-alpha (TNF-alpha) and leukotrienes by AM from the GM(-/-) mice was greatly reduced compared with wild-type AM, whereas secretion of monocyte chemoattractant protein-1 was increased. Transgenic expression of GM-CSF exclusively in the lungs of GM(-/-) mice resulted in AM with normal or supranormal expression of CD11a and CD11c, phagocytic activity, and TNF-alpha secretion. Thus, in the absence of GM-CSF, AM functional capabilities for host defense were significantly impaired but were restored by lung-specific expression of GM-CSF.     

Death of the Escherichia coli K-12 strain W3110 in soil and water.

Whether Escherichia coli K-12 strain W3110 can enter the "viable but nonculturable" state was studied with sterile and nonsterile water and soil at various temperatures. In nonsterile river water, the plate counts of added E. coli cells dropped to less than 10 CFU/ml in less than 10 days. Acridine orange direct counts, direct viable counts, most-probable-number estimates, and PCR analyses indicated that the added E. coli cells were disappearing from the water in parallel with the number of CFU. Similar results were obtained with nonsterile soil, although the decline of the added E. coli was slower. In sterile water or soil, the added E. coli persisted for much longer, often without any decline in the plate counts even after 50 days. In sterile river water at 37 degrees C and sterile artificial seawater at 20 and 37 degrees C, the plate counts declined by 3 to 5 orders of magnitude, while the acridine orange direct counts remained unchanged. However, direct viable counts and various resuscitation studies all indicated that the nonculturable cells were nonviable. Thus, in either sterile or nonsterile water and soil, the decline in plate counts of E. coli K-12 strain W3110 is not due to the cells entering the viable but nonculturable state, but is simply due to their death.