Control of heterotypic fibril formation by collagen V is determined by chain stoichiometry.

Although the collagen V heterotrimer is known to be involved in the control of fibril assembly, the role of the homotrimer in fibrillar organization has not yet been examined. Here, the production of substantial amounts of recombinant collagen V homotrimer has allowed a detailed study of its role in homotypic and heterotypic fibril formation. After removal of terminal regions by pepsin digestion, both the collagen V heterotrimer and homotrimer formed thin homotypic fibrils, thus showing that diameter limitation is at least in part an intrinsic property of the collagen V triple helix. When mixed with collagen I, however, various complementary approaches indicated that the collagen V heterotrimer and homotrimer exerted different effects in heterotypic fibril formation. Unlike the heterotrimer, which was buried in the fibril interior, the homotrimer was localized as thin filamentous structures at the surface of wide collagen I fibrils and did not regulate fibril assembly. Its localization at the fibril surface suggests that the homotrimer can act as a molecular linker between collagen fibrils or macromolecules in the extracellular matrix or both. Thus, depending on their respective distribution in tissues, the different collagen V isoforms might fulfill specific biological functions.     

Distinct maturations of N-propeptide domains in fibrillar procollagen molecules involved in the formation of heterotypic fibrils in adult sea urchin collagenous tissues

We have characterized the primary structure of a new sea urchin fibrillar collagen, the 5alpha chain, including nine repeats of the sea urchin fibrillar module in its N-propeptide. By Western blot and immunofluorescence analyses, we have shown that 5alpha is co-localized in adult collagenous ligaments with the 2alpha fibrillar collagen chain and fibrosurfin, two other extracellular matrix proteins possessing sea urchin fibrillar modules. At the ultrastructural level, the 5alpha N-propeptide is detected at the surface of fibrils, suggesting the retention of this domain in mature collagen molecules. Biochemical characterization of pepsinized collagen molecules extracted from the test tissue (the endoskeleton) together with a matrix-assisted laser desorption ionization time-of-flight analysis allowed us to determine that 5alpha is a quantitatively minor fibrillar collagen chain in comparison with the 1alpha and 2alpha chains. Moreover, 5alpha forms heterotrimeric molecules with two 1alpha chains. Hence, as in vertebrates, sea urchin collagen fibrils are made up of quantitatively major and minor fibrillar molecules undergoing distinct maturation of their N-propeptide regions and participating in the formation of heterotypic fibrils.     

Rat RAMP domains involved in adrenomedullin binding specificity

When coexpressed with receptor activity-modifying protein (RAMP)2 or -3, calcitonin receptor-like receptor (CRLR) functions as an adrenomedullin (AM) receptor (CRLR/RAMP2 or -3). Coexpression of rat (r)CRLR with rRAMP deletion mutants in HEK293T cells revealed that deletion of residues 93-99 from rRAMP2 or residues 58-64 from rRAMP3 significantly inhibits high-affinity [125I]AM binding and AM-evoked cAMP production, despite full cell surface expression of the receptor heterodimer. Apparently, these two seven-residue segments are key determinants of high-affinity agonist binding to rAM receptors and of receptor functionality. Consequently, their deletion yields peptides that are able to serve as negative regulators of AM receptor function.     

Analysis of functional domains of the host cell factor involved in VP16 complex formation.

We present biochemical analyses of the regions of the host cell factor (HCF) involved in VP16 complex formation and in the association between the N- and C-terminal domains of HCF itself. We show that the kelch repeat region of HCF (residues 1-380) is sufficient for VP16 complex formation, but that residues C-terminal to the repeats (positions 381-450) interfere with this activity. However, these latter residues are required for the interaction between the N- and C-terminal regions of HCF. The extreme C-terminal region of HCF, corresponding to an area of strong conservation with a Caenorhabditis elegans homologue, is sufficient for interaction with the N-terminal region. These results are discussed with respect to possible differences in the roles of HCF in VP16 activity versus its normal cellular function.     

Mapping EBNA-1 domains involved in binding to metaphase chromosomes

The Epstein-Barr virus (EBV) genome can persist in dividing human B cells as multicopy circular episomes. Viral episomes replicate in synchrony with host cell DNA and are maintained at a relatively constant copy number for a long time. Only two viral elements, the replication origin OriP and the EBNA-1 protein, are required for the persistence of viral genomes during latency. EBNA-1 activates OriP during the S phase and may also contribute to the partition and/or retention of viral genomes during mitosis. Indeed, EBNA-1 has been shown to interact with mitotic chromatin. Moreover, viral genomes are noncovalently associated with metaphase chromosomes. This suggests that EBNA-1 may facilitate the anchorage of viral genomes on cellular chromosomes, thus ensuring proper partition and retention. In the present paper, we have investigated the chromosome-binding activity of EBV EBNA-1, herpesvirus papio (HVP) EBNA-1, and various derivatives of EBV EBNA-1, fused to a variant of the green fluorescent protein. The results show that binding to metaphase chromosomes is a common property of EBV and HVP EBNA-1. Further studies indicated that at least three independent domains (CBS-1, -2, and -3) mediate EBNA-1 binding to metaphase chromosomes. In agreement with the anchorage model, two of these domains mapped to a region that has been previously demonstrated to be required for the long-term persistence of OriP-containing plasmids.     

Mutational analysis of protein binding sites involved in formation of the bacteriophage lambda attL complex.

Bacteriophage lambda site-specific recombination requires the formation of higher-order protein-DNA complexes to accomplish synapsis of the partner attachment (att) sites as well as for the regulation of the integration and excision reactions. The att sites are composed of a core region, the actual site of strand exchange, and flanking arm regions. The attL site consists of two core sites (C and C'), an integration host factor (IHF) binding site (H'), and three contiguous Int binding arm sites (P'1, P'2, and P'3). In this study, we employed bacteriophage P22 challenge phages to determine which protein binding sites participate in attL complex formation in vivo. The C', H', and P'1 sites were critical, because mutations in these sites severely disrupted formation of the attL complex. Mutations in the C and P'2 sites were less severe, and alteration of the P'3 site had no effect on complex formation. These results support a model in which IHF, bound to the H' site, bends the attL DNA so that the Int molecule bound to P'1 also interacts with the C' core site. This bridged complex, along with a second Int molecule bound to P'2, helps to stabilize the interaction of a third Int with the C core site. The results also indicate that nonspecific DNA binding is a significant component of the Int-core interactions and that the cooperativity of Int binding can overcome the effects of mutations in the individual arm sites and core sites.     

Localization of the domains of fibrin involved in binding to platelets

The molecular basis of platelet-fibrin interactions has been investigated by using synthetic peptides as potential inhibitors of fibrin protofibril and fibrinogen binding to ADP-stimulated platelets, adhesion of fibrin fibers to the platelet surface, and platelet-mediated clot retraction. Synthetic peptides of sequence RGDS and HHLGGAKQAGDV, corresponding to regions of the fibrinogen alpha- and gamma-chains previously identified as platelet recognition sites, inhibited the binding of radiolabelled soluble fibrin oligomers to ADP-stimulated platelets with IC50 values of 10 and 40 microM, respectively. Synthetic GPRP and GHRP, corresponding to the N-terminal tripeptide sequence of the fibrin alpha-chains and the tetrapeptide sequence of the beta-chains, respectively, were minimally effective in blocking soluble fibrin polymer binding to ADP-stimulated platelets. Platelet functions which are unique to the three-dimensional fibrin network were examined by measurements of the extent of adhesion of fluorophore-labelled fibrin to platelets with a microfluorimetric technique and by light scattering measurements of the time course of clot retraction. Inhibition of fibrin-platelet adhesion by RGDS, HHLGGAKQAGDV and GHRP exhibited a similar, linear dependence reaching 1/2 maximum at about 200 microM, suggesting nonspecific effects. GPRP inhibited fibrin assembly but did not appear to have specific effects on fibrin-platelet adhesion. Only RGDS effected clot retraction, causing a 4-6-fold decrease in rate at 230 microM. These results indicate that fibrinogen and fibrin protofibrils, which are obligatory intermediates on the fibrin assembly pathway, share a set of common platelet recognition sites located at specific regions of the alpha- and gamma-chains of the multinodular fibrin(ogen) molecules. The RGDS site is also involved in mediating interactions between the three-dimensional fibrin network and stimulated platelets.     

The aminoacyl-tRNA synthetase-tRNA complex: detection by differential labelling of lysine residues involved in complex formation

The interaction of tRNA^Tyr with tyrosyl-tRNA synthetase from Bacillus stearothermophilus was studied by differential acetylation of lysine residues. The synthetase was trace-labelled in the free form and as the synthetase-tRNA^Tyr complex with [³H]acetic anhydride. In a second step the two ³H-labelled enzyme preparations were fully acetylated with cold reagent under denaturing conditions and were mixed with synthetase that had been homogeneously labelled with excess [¹⁴C]acetic anhydride. Peptides containing labelled lysine residues were isolated after chymotryptic digestion and their ${}^{14}\text{C}{}^3\text{H}$ ratios were determined. These ratios reflect the reactivity of primary amino groups towards acetic anhydride.Involvement of lysine side-chains in complex formation with tRNA^Tyr was suggested from altered ${}^{14}\text{C}{}^3\text{H}$ ratios. Out of the 22 primary amino groups of tyrosyl-tRNA synthetase at least three showed reduced reactivities towards acetic anhydride in the synthetase-tRNA^Tyr complex by factors of 1.6, 1.9 and 6.8, respectively. The sequences around these lysine residues have been determined enabling their placement when the primary and tertiary structure of the enzyme are available (G. L. E. Koch, to be published). No lysine residue of increased reactivity in the synthetase-tRNA^Tyr complex has been detected.Only one molecule of tRNA^Tyr binds to the dimeric synthetase molecule under the conditions of the differential labelling. If the binding site for the tRNA is on one of the two identical subunits, any observed decrease in chemical reactivity of a particular lysine residue should not exceed a factor of two. The detection of a lysine residue which reacts about seven times more slowly in the synthetase-tRNA complex could therefore indicate that the single binding site is formed by both enzyme subunits.     

Ligand binding by TPR domains

Tetratricopeptide repeat (TPR) domains bind specific peptide ligands and are thought to mediate protein-protein interactions in a variety of biological systems. Here we compare peptide ligand-binding by several different TPR domains. We present specific examples that demonstrate that TPR domains typically undergo little or no structural rearrangement upon ligand binding. Our data suggest that, contrary to a recent proposal, coupled folding and binding is not the common mechanism of ligand recognition by TPR domains.     

Controlling cell position in complex heterotypic 3D microtissues by tissue fusion

Tissue fusion and cell sorting are processes fundamental to developmental biology with applications in tissue engineering. We have designed a fusion assay to investigate the factors governing the fusion of microtissues and the cell sorting that occurs after fusion. Normal human fibroblast (NHF) spheroids were self‐assembled and cultured for 1, 4, or 7 days, then combined in trough shaped recesses. Over a 24‐h period the spheroids fused to become a rod shaped microtissue and the kinetics and extent of fusion could be quantified by assessing rod contraction. By varying the amount of spheroid culture time prior to fusion (1–7 days), the rate of fusion, the coherence of the building units (as measured by fusion angle) and the steady state length of the structure could be easily controlled. Longer pre‐culture times for the spheroids resulted in slower fusion, less coherence and increased length of rod microtissues. The fusion kinetics and steady length of rods formed by smaller versus larger spheroids (～100 vs. 300 µm diameter) were indistinguishable, even though smaller spheroids had twice the surface area and greater numbers of contacts between units. Both small and large spheroids were strongly influenced by spheroid pre‐culture time. Pre‐culture time could also be used to control cell sorting and cell position when combinations of NHFs and H35s, a rat hepatocyte cell line, were fused to form heterotypic microtissues. Control of fusion and cell position are important parameters for creating functional heterotypic microtissues as well as the use of microtissues as building units to create larger tissue structures. Biotechnol. Bioeng. 2009;102: 1231–1241. © 2008 Wiley Periodicals, Inc.     

DNA repeated sequences may be involved in synaptonemal complex formation

Synaptonemal complexes (SCs) are intranuclear structures that facilitate the reversible lateral synapsis of homologous chromosomes in the course of meiosis. It is still unclear which DNA nucleotide sequences are responsible for the attachment of chromatin to SC lateral elements. Considering the features of the dispersed repeated sequences (RSs), it is possible to assume that they participate in the structure and functional organization of the meiotic chromosomes. Using numerical analysis, we have investigated the relationship between the RS and the distribution of meiotic recombination events in mouse chromosome 1. Using in situ hybridization on spread mouse spermatocytes, we have examined the arrangement of different types of RSs relative to SCs. Hybridization signals of B1(Alu), B2, and minisatellite probes were localized predominantly in SCs regions. Based on the results, we proposed a model of meiotic chromosome organization. According to the model, RSs participate in the attachment of chromatin loops to SCs.     

Peptide binding domains determined through chemical modification of the side-chain functional groups.

A clear understanding of the specific secondary structure and binding domain resulting from the interactions of proteins and peptides with lipid surfaces will provide insight into the specific functions of biologically active molecules. We have shown in earlier studies that the stationary phases used in reverse-phase high-performance liquid chromatography represent a model artificial lipid surface for the study of induced conformational states of peptides on lipid interaction. We have now used reverse-phase high-performance liquid chromatography to determine the binding domains of peptides and, by extension, of proteins to a lipid surface. This approach consists of performing chemical modifications of specific amino acid side-chain functionalities after the interaction of the peptides with the reverse-phase high-performance liquid chromatography C18 groups. The susceptibility to oxidation was also studied after binding of the same peptides to liposomes. Oxidation of a single methionine residue "walked" through an amphipathic alpha-helical 18-mer peptide was selected to illustrate this approach. The extent of oxidation was found to be clearly dictated by the accessibility of the methionine residue to the aqueous mobile phase. The binding domain found for the peptide in its lipid-induced conformational state was unequivocally the entire hydrophobic face of the amphipathic alpha-helix.     

Phosphorus bioavailability of fluvial sediments determined by algal assays

Eroding bank soils and riverine suspended sediments from the Flathead River-Lake ecosystem, Montana, USA, were cultured with the alga Selenastrum capricornutum Printz in PAAP medium with the sediments as the sole source of phosphorus. Extraction of phosphorus by NaOH and nitrilotriacetic acid (NTA) solutions were compared to results from algal bioassays. The fine sediment particles transported into Flathead Lake during spring runoff had the highest availability (i.e. 6% of total phosphorus). Bank soils which contained the greatest percentage of fine clays exhibited similar (i.e. 4% of total phosphorus) availability. Bank soils containing predominantly organic phosphorus had the lowest availability. Spearman's rank correlation indicated significance at the 5% test level between algal assay estimates of available phosphorus and both chemical extraction techniques.     

Thermostability of allicin determined by chemical and biological assays

The garlic-derived antibacterial principle, alk(en)yl sulfinate compounds, has long been considered as very short-lived substance. However, there are some data showing a rather more stable nature of allicin. We determined here the thermostability of allicin by a systematic analyses employing chemical quantification and an antibacterial activity assay. Allicin in an aqueous extract of garlic was degraded stoichiometrically in proportion to the temperature; we estimated the half-life of allicin to be about a year at 4 degrees C (from 1.8 mg/ml to 0.9 mg/ml) and 32 d at 15 degrees C, but only 1 d at 37 degrees C (from 2.0 mg/ml to 1.0 mg/ml). The half-life values for antibacterial activity showed a similar trend in results: 63 d or more at 4 degrees C for both antibacterial activities, 14 d for anti-staphylococcal activity, and 26 d for anti-escherichia activity at 15 degrees C, but only 1.2 d and 1.9 d for the respective activities at 37 degrees C. Such antibacterial activities were attributable to the major allicin, allyl 2-propenylthiosulfinate. Surprisingly, the decline in the quantity of allicin was not accompanied by its degradation; instead, allicin became a larger molecule, ajoene, which was 3-times larger than allicin.     

HURP is part of a Ran-dependent complex involved in spindle formation

BACKGROUND: GTP-loaded Ran induces the assembly of microtubules into aster-like and spindle-like structures in Xenopus egg extract. The microtubule-associated protein (MAP), TPX2, can mediate Ran's role in aster formation, but factors responsible for the transition from aster-like to spindle-like structures have not been described. RESULTS: Here we identify a complex that is required for the conversion of aster-like to spindle-like structures. The complex consists of two characterized MAPs (TPX2, XMAP215), a plus end-directed motor (Eg5), a mitotic kinase (Aurora A), and HURP, a protein associated with hepatocellular carcinoma. Formation and function of the complex is dependent on Aurora A activity. HURP protein was further characterized and shown to bind microtubules and affect their organization both in vitro and in vivo. In egg extract, anti-HURP antibodies disrupt the formation of both Ran-dependent and chromatin and centrosome-induced spindles. HURP is also required for the proper formation and function of mitotic spindles in HeLa cells. CONCLUSIONS: HURP is a new and essential component of the mitotic apparatus. HURP acts as part of a multicomponent complex that affects the growth or stability of spindle MTs and is required for spindle MT organization.     

Calcium binding domains of calmodulin. Sequence of fill as determined with terbium luminescence

Terbium, a trivalent lanthanide, effectively substituted for Ca2+ in calmodulin as judged by several criteria: intrinsic fluorescence spectra, altered mobilities on polyacrylamide gel electrophoresis, formation of a stable complex with troponin I or calcineurin, and stimulation of phosphodiesterase. Calmodulin harbors four Ca2+ binding domains; domains I and II contain no tyrosine, whereas domains III and IV each have one tyrosine. The binding of Tb3+ to calmodulin was followed by the increase of Tb3+ fluorescence at 545 nm upon binding to calmodulin. This fluorescence was elicited either by exciting Tb3+ directly at 222 nm or by exciting the calmodulin tyrosine at 280 nm with resulting energy transfer from tyrosine to Tb3+. Fluorescence generated by direct excitation measures binding of Tb3+ to any of the Ca2+ binding domains, whereas energy transfer through indirect excitation is effective only when Tb3+ is within 5 A of tyrosine, indicating that Tb3+ necessarily occupies a Ca2+ binding domain that contains tyrosine. A judicious use of the direct and indirect excitation could reveal the sequence of fill of the binding domains. Our results suggest these domains are filled in the following sequence: 1) domain I or II; 2) domains III and IV; and 3) domain II or I that has not been filled initially.     

Global phylogeny determined by the combination of protein domains in proteomes

The majority of proteins consist of multiple domains that are either repeated or combined in defined order. In this study, we survey the combination of protein domains defined at fold and fold superfamily levels in 185 genomes belonging to organisms that have been fully sequenced and introduce a method that reconstructs rooted phylogenomic trees from the content and arrangement of domains in proteins at a genomic level. We find that the majority of domain combinations were unique to Archaea, Bacteria, or Eukarya, suggesting most combinations originated after life had diversified. Domain repeat and domain repeat within multidomain proteins increased notably in eukaryotes, mainly at the expense of single-domain and domain-pair proteins. This increase was mostly confined to Metazoa. We also find an unbalanced sharing of domain combinations which suggests that Eukarya is more closely related to Bacteria than to Archaea, an observation that challenges the widely assumed eukaryote-archaebacterial sisterhood relationship. The occurrence and abundance of the molecular repertoire (interactome) of domain combinations was used to generate phylogenomic trees. These global interactome-based phylogenies described organismal histories satisfactorily, revealing the tripartite nature of life, and supporting controversial evolutionary patterns, such as the Coelomata hypothesis, the grouping of plants and animals, and the Gram-positive origin of bacteria. Results suggest strongly that the process of domain combination is not random but curved by evolution, rejecting the null hypothesis of domain modules combining in the absence of natural selection or an optimality criterion.