Self-association of IQGAP1: characterization and functional sequelae

The scaffolding protein IQGAP1 participates in numerous cellular functions by binding to target proteins such as actin, calmodulin, E-cadherin, beta-catenin, Cdc42, Rac1, and CLIP-170. IQGAP1 regulates the cytoskeleton, promotes cell motility, and modulates E-cadherin-mediated cell-cell adhesion. However, how IQGAP1 exerts its functions in vivo is still unclear. In this study we investigate the self-association of IQGAP1 and its role in IQGAP1 function. Endogenous IQGAP1 co-immunoprecipitated from MCF-7 cells with IQGAP1 tagged with enhanced green fluorescent protein, indicating that IQGAP1 self-associates in cells. In vitro assays confirmed that IQGAP1 can self-associate and that this effect is mediated by the N-terminal half of the protein. Gel filtration analysis suggested that full-length IQGAP1 exists as a combination of monomers, dimers, and larger oligomers. Analysis performed with multiple fragments of IQGAP1 narrowed the self-association region to amino acids 763-863. In support of this observation, a peptide comprising residues 763-863 disrupted self-association of full-length IQGAP1 in a dose-dependent manner. Similarly, deleting this sequence from IQGAP1 abolished binding to full-length IQGAP1. In addition, the ability of IQGAP1 to increase the amount of active Cdc42 in cells is abrogated upon removal of this region. Consistent with these findings, transfection into cells of a peptide containing the self-association domain significantly reduced the amount of active Cdc42 in cell lysates. These observations define a sequence of IQGAP1 that is necessary for its oligomerization and demonstrate that self-association is required for the normal cellular function of IQGAP1.     

Forecasting phenology: from species variability to community patterns

Shifts in species' phenology in response to climate change have wide-ranging consequences for ecological systems. However, significant variability in species' responses, together with limited data, frustrates efforts to forecast the consequences of ongoing phenological changes. Herein, we use a case study of three North American plant communities to explore the implications of variability across levels of organisation (within and among species, and among communities) for forecasting responses to climate change. We show how despite significant variation among species in sensitivities to climate, comparable patterns emerge at the community level once regional climate drivers are accounted for. However, communities differ with respect to projected patterns of divergence and overlap among their species' phenological distributions in response to climate change. These analyses and a review of hypotheses suggest how explicit consideration of spatial scale and levels of biological organisation may help to understand and forecast phenological responses to climate change.     

A glycosylated form of the human cardiac hormone pro B-type natriuretic peptide is an intrinsically unstructured monomeric protein

The N-terminal fragment of pro B-type natriuretic peptide (NT-proBNP) and proBNP are used as gold standard clinical markers of myocardial dysfunction such as cardiac hypertrophy and left ventricle heart failure. The actual circulating molecular forms of these peptides have been the subject of intense investigation particularly since these analytes are measured in clinical assays. Conflicting data has been reported and no firm consensus on the exact nature of the molecular species exists. Because these clinical assays are immunoassay-based, specific epitopes are detected. It is conceivable then that certain epitopes may be masked and therefore unavailable for antibody binding, thus the importance of determining the nature of the circulating molecular forms of these analytes. This situation is an unavoidable Achilles’ heel of immunoassays in general.A recombinant O-linked glycosylated form of proBNP has been show to mimic some of the properties of extracted plasma from a heart failure patient. In particular the recombinant and native material co-migrated as diffuse Western-immunostained bands on SDS-PAGE and each band collapsed to an apparent homogeneous band following deglycosylation. Thus, glycosylated-proBNP may be one such circulating form. Here we provide extensive physiochemical characterization for this O-linked protein and compare these results to other described circulating species, non-glycosylated-proBNP and NT-proBNP. It will be shown that glycosylation has no influence on the secondary and quaternary structure of proBNP. In fact, at moderate concentration in benign physiological neutral pH buffer, all three likely circulating species are essentially devoid of major secondary structure, i.e., are intrinsically unstructured proteins (IUPs). Furthermore, all three proteins exist as monomers in solution. These results may have important implications in the design of NT-proBNP/BNP immunoassays.     

Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. II. IgG antibodies contain VH genes from a single VH family and VL genes from at least four VL families

To define the V gene family repertoire of human IgG anti-Haemophilus influenzae type b polysaccharide antibodies, we purified six IgG1 and nine IgG2 anti-Hib-PS antibodies to monoclonality from immune serum of six individuals and performed N-terminal amino acid sequence analysis. Of the 15 clonal antibodies we examined, all H chain V regions were of the VHIII family. In contrast, the L chains of these antibodies were clearly from at least four different VL families; VKI, VKII, VKIII, and V lambda. Interestingly. VL family expression correlated with the cross-reactivity of these antibodies to the capsular carbohydrate of Escherichia coli K100. VKII antibodies did not cross-react, whereas antibodies expressing V lambda, VKI, or VKIII generally cross-reacted. We conclude that L chain V regions are very important contributors to the limited heterogeneity in this antibody repertoire.     

The role of human ribosomal proteins in the maturation of rRNA and ribosome production

Production of ribosomes is a fundamental process that occurs in all dividing cells. It is a complex process consisting of the coordinated synthesis and assembly of four ribosomal RNAs (rRNA) with about 80 ribosomal proteins (r-proteins) involving more than 150 nonribosomal proteins and other factors. Diamond Blackfan anemia (DBA) is an inherited red cell aplasia caused by mutations in one of several r-proteins. How defects in r-proteins, essential for proliferation in all cells, lead to a human disease with a specific defect in red cell development is unknown. Here, we investigated the role of r-proteins in ribosome biogenesis in order to find out whether those mutated in DBA have any similarities. We depleted HeLa cells using siRNA for several individual r-proteins of the small (RPS6, RPS7, RPS15, RPS16, RPS17, RPS19, RPS24, RPS25, RPS28) or large subunit (RPL5, RPL7, RPL11, RPL14, RPL26, RPL35a) and studied the effect on rRNA processing and ribosome production. Depleting r-proteins in one of the subunits caused, with a few exceptions, a decrease in all r-proteins of the same subunit and a decrease in the corresponding subunit, fully assembled ribosomes, and polysomes. R-protein depletion, with a few exceptions, led to the accumulation of specific rRNA precursors, highlighting their individual roles in rRNA processing. Depletion of r-proteins mutated in DBA always compromised ribosome biogenesis while affecting either subunit and disturbing rRNA processing at different levels, indicating that the rate of ribosome production rather than a specific step in ribosome biogenesis is critical in patients with DBA.     

Onset of summer flowering in a 'Sky Island' is driven by monsoon moisture

Temperatures for the southwestern USA are predicted to increase in coming decades, especially during the summer season; however, little is known about how summer precipitation patterns may change. We aimed to better understand how nonsucculent plants of a water-limited gradient encompassing xeric desert to mesic mountain-top may respond to changes in summer conditions. We used a species-rich 26-yr flowering record to determine species' relationships with precipitation and temperature in months coincident with and previous to flowering. The onset of summer flowering was strongly influenced by the amount and timing of July precipitation, regardless of elevation or life form, suggesting the critical importance of soil moisture in triggering summer flowering in this region. Future changes in the timing or consistency of the early monsoon will probably impact directly on the onset of flowering for many species in this region. In addition, a key implication of predicted increasing temperatures is a decrease in available soil moisture. At all elevations, many species may be expected to flower later in the summer under the decreased soil moisture conditions associated with warmer temperatures. However, impacts on summer flowering may be greater at higher elevations, because of the greater sensitivity of mesic plants to water stress.     

A 68 residue N-terminal fragment of pro-atrial natriuretic peptide is a monomeric intrinsically unstructured protein

The mature pro forms of the cardiac natriuretic peptides, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), are proteolytically processed to their active hormone forms (28 and 32 residues, respectively) and N-terminal (NT)-pro fragments (68 and 76 residues, respectively). Far-ultraviolet circular dichroism (UV CD), 1D and 2D-homonuclear nuclear magnetic resonance (NMR), size exclusion-high performance liquid chromatography (SE-HPLC) and analytical ultracentrifuge sedimentation equilibrium (AUCSE) data are obtained for NT-proANP. CD data showed a large negative molar ellipticity for NT-proANP of -14,800° cm(2)/dmol at 199-200 nm. The intensity of the 1D-(1)H NMR spectra in the amide region for NT-proANP was very low and confined to ~8-8.6 ppm. Furthermore, cross-correlation resonance peaks were absent in the corresponding 2D-(1)H NOE spectra for NT-proANP in this region. The elution peak for this fragment from a G2000SW size-exclusion column was 20.4'; myoglobin (~17 K) was also eluted at 20.4'. No higher molecular weight oligomers were evident in the AUCSE experiments for NT-proANP. Collectively, the physical data demonstrate that NT-proANP, like NT-proBNP, is primarily a disordered, monomeric protein. Lastly, we compare the predictions from two in silico metaserver disorder algorithms, MeDor and MetaPrDOS, to the experimental data.     

The sites in the I-Ak histocompatibility molecule photoaffinity labeled by an immunogenic lysozyme peptide

The class II histocompatibilty molecule I-Ak was photoaffinity labeled by NH2- and COOH-terminal photoreactive conjugates of an immunogenic hen egg white lysozyme (HEL) peptide. The labeled alpha and beta chains were digested with protease from Staphylococcus aureus strain V-8 (protease V-8) and/or trypsin, and the proteolytic fragments were separated by high performance liquid chromatography (HPLC) (peptide mapping). Reproducible peptide maps containing a major labeled component were obtained from the three conjugates reported here whose photoreactive group was attached via short spacers of limited flexibility. The COOH-terminal conjugate N-acetyl HEL-(49-61)-iodo-4-azidosalicyloyl thioester (compound 1) labeled hydrophilic tryptic digest fragments on both chains of I-Ak. The labeled digest fragments were homogeneous in reverse-phase and anion-exchange HPLC, indicating that the photoaffinity labeling was site-specific. Conversely, the NH2-terminal conjugate iodo-4-azidosalicyloyl HEL-(46-61) (compound 2: IASA-(46-61)) labeled exceptionally hydrophobic sequences on both chains of I-Ak. The labeling was also site-specific because reverse-phase HPLC of primary digests with protease V-8 and secondary digests with trypsin showed single major labeled components. The labeling of I-Ak by IASA-(46-61) was fully inhibitible by HEL-(46-61). In contrast, IASA attached to the smallest immunogenic peptide 52-61 (compound 3) labeled a distinctly different hydrophilic tryptic fragment. The site of the I-Ak molecule that was photoaffinity labeled by IASA-(46-61) (compound 2) was determined. IASA-(46-61) labeled selectively at Pro-118 of a primary alpha chain fragment most likely encompassing residues 115-134. It labeled Thr-121 of a primary beta chain fragment most likely encompassing residues 109-138. We also obtained evidence that IASA-(46-61) occupied the antigen-specific site; the conjugate stimulated a T-cell hybridoma that recognizes the sequence 52-61 and also competed for the binding of this smaller peptide to I-Ak. Thus, peptides that bind to the allele-specific binding site and are long enough to extend beyond it can interact with a hydrophobic area of class II molecules. This area is formed by sequences of the first halves of the second domain of both alpha and beta chains.     

Utility of the white gene in estimating phylogenetic relationships among mosquitoes (Diptera: Culicidae)

The utility of a nuclear protein-coding gene for reconstructing phylogenetic relationships within the family Culicidae was explored. Relationships among 13 species representing three subfamilies and nine genera of Culicidae were analyzed using a 762-bp fragment of coding sequence from the eye color gene, white. Outgroups for the study were two species from the sister group Chaoboridae. Sequences were determined from clone PCR products amplified from genomic DNA, and aligned following conceptual intron splicing and amino acid translation. Third codon positions were characterized by high levels of divergence and biased nucleotide composition, the intensity and direction of which varied among taxa. Equal weighting of all characters resulted in parsimony and neighboring-joining trees at odds with the generally accepted phylogenetic hypothesis based on morphology and rDNA sequences. The application of differential weighting schemes recovered the traditional hypothesis, in which the subfamily Anophelinae formed the basal clade. The subfamily Toxorhynchitinae occupied an intermediate position, and was a sister group to the subfamily Culicinae. Within Culicinae, the genera Sabethes and Tripteroides formed an ancestral clade, while the Culex-Deinocerites and Aedes- Haemagogus clades occupied increasingly derived positions in the molecular phylogeny. An intron present in the Culicinae- Toxorhynchitinae lineage and one outgroup taxon was absent in the basal Anophelinae lineage and the second outgroup taxon, suggesting that intron insertions or deletions may not always be reliable systematic characters.      

Relating mesocarnivore relative abundance to anthropogenic land‐use with a hierarchical spatial count model

There is growing need to develop models of spatial patterns in animal abundance, yet comparatively few examples of such models exist. This is especially true in situations where the abundance of one species may inhibit that of another, such as the intensively‐farmed landscape of the Prairie Pothole Region (PPR) of the central United States, where waterfowl production is largely constrained by mesocarnivore nest predation. We used a hierarchical Bayesian approach to relate the distribution of various land‐cover types to the relative abundances of four mesocarnivores in the PPR: coyote Canis latrans, raccoon Procyon lotor, red fox Vulpes vulpes, and striped skunk Mephitis mephitis. We developed models for each species at multiple spatial resolutions (41.4 km², 10.4 km², and 2.6 km²) to address different ecological and management‐related questions. Model results for each species were similar irrespective of resolution. We found that the amount of row‐crop agriculture was nearly ubiquitous in our best models, exhibiting a positive relationship with relative abundance for each species. The amount of native grassland land‐cover was positively associated with coyote and raccoon relative abundance, but generally absent from models for red fox and skunk. Red fox and skunk were positively associated with each other, suggesting potential niche overlap. We found no evidence that coyote abundance limited that of other mesocarnivore species, as might be expected under a hypothesis of mesopredator release. The relationships between relative abundance and land‐cover types were similar across spatial resolutions. Our results indicated that mesocarnivores in the PPR are most likely to occur in portions of the landscape with large amounts of agricultural land‐cover. Further, our results indicated that track‐survey data can be used in a hierarchical framework to gain inferences regarding spatial patterns in animal relative abundance.