Finding functional sites in structural genomics proteins

Assigning function to structures is an important aspect of structural genomics projects, since they frequently provide structures for uncharacterized proteins. Similarities uncovered by structure alignment can suggest a similar function, even in the absence of sequence similarity. For proteins adopting novel folds or those with many functions, this strategy can fail, but functional clues can still come from comparison of local functional sites involving a few key residues. Here we assess the general applicability of functional site comparison through the study of 157 proteins solved by structural genomics initiatives. For 17, the method bolsters confidence in predictions made based on overall fold similarity. For another 12 with new folds, it suggests functions, including a putative phosphotyrosine binding site in the Archaeal protein Mth1187 and an active site for a ribose isomerase. The approach is applied weekly to all new structures, providing a resource for those interested in using structure to infer function.     

Enzymatic synthesis of tea theaflavin derivatives and their anti-inflammatory and cytotoxic activities

Derivatives based on a benzotropolone skeleton (9-26) have been prepared by the enzymatic coupling (horseradish peroxidase/H2O2) of selected pairs of compounds (1-8), one with a vic-trihydroxyphenyl moiety, and the other with an ortho-dihydroxyphenyl structure. Some of these compounds have been found to inhibit TPA-induced mice ear edema, nitric oxide (NO) synthesis, and arachidonic acid release by LPS-stimulated RAW 264.7 cells. Their cytotoxic activities against KYSE 150 and 510 human esophageal squamous cell carcinoma and HT 29 human colon cancer cells were also evaluated.     

Expression of Trisk 51, agrin and nicotinic-acetycholine receptor ε-subunit during muscle development in a novel three-dimensional muscle-neuronal co-culture system

The purpose of our study was to create functional muscle tissue in vitro and to investigate the influence of organotypic neuronal slice cultures from rat spinal cord on the differentiation and function of primary rat myoblasts in a novel three-dimensional culture system. Three-dimensional muscle-neuronal cultures were established by co-cultivating primary rat skeletal muscle cells of newborn rats with organotypic slice cultures of the spinal cord prepared from isogenic rats in a fibrin matrix. These constructs were cultured for up to 4 weeks. Differentiation and fusion of the myoblasts to myofibers was evaluated by analyzing the expression pattern and localization of muscle- and neuron-specific markers. The fibrin matrix provided a suitable environment for three-dimensional myoblast culture. Co-culturing of organotypic spinal cord slices with myoblasts induced the formation of spontaneously contracting multinuclear and parallel-aligned myofibers. Pharmacological tests suggested the formation of neuromuscular junctions. The analysis of neural agrin expression and myogenic desmin, myogenin, MyoD, Trisk 51, and nicotinic-acetycholine receptor (nACh-receptor) -subunit expression revealed the differentiation of the myoblasts to myofibers. The presented novel three-dimensional co-culture system allows the in vitro investigation of myoblast differentiation and neuron-myoblast interaction. Our results suggest the existence of an alternative pathway for the maturation of the nAChR -subunit to the -subunit without neural agrin activity.A.D.B. and J.P.B. contributed equally to this studyThis work was supported by a major grant from the State of Baden-Württemberg within the scope of the Valley TEC (Valley Tissue Engineering Center)     

Imidazole derivatives as a novel class of hybrid compounds with inhibitory histamine N-methyltransferase potencies and histamine hH3 receptor affinities

In this study, a novel series of imidazole-containing compounds with dual properties, that is, inhibitory potency at the enzyme histamine N(tau)-methyltransferase (HMT) and antagonist potency at histamine H(3) receptors was designed and synthesized. Pharmacologically, these new hybrid drugs were evaluated in functional assays for their inhibitory potencies at rat kidney HMT and for their antagonist activities on synaptosomes of rat cerebral cortex. For selected compounds, binding affinities at recombinant human histamine H(3) receptors were determined. The first compounds (1-10) of the series proved to be H(3) receptor ligands of high potency at rat synaptosomes or of high binding affinity at human H(3) receptors, respectively, but of only moderate activity as inhibitors of rat kidney HMT. In contrast, aminoquinoline- or tetrahydroacridine-containing derivatives 11-17 also displayed HMT inhibitory potency in the nanomolar concentration range. Preliminary data from molecular modeling investigations showed that the imidazole derivative 15 and the HMT inhibitor quinacrine possess identical binding areas. The most interesting compound (14) is simultaneously a highly potent H(3) receptor ligand (K(i)=4.1nM) and a highly potent HMT inhibitor (IC(50)=24nM), which makes this derivative a valuable pharmacological tool for further development.     

The in vitro and in vivo effects of anti-galactose antibodies on endothelial cell activation and xenograft rejection

We have previously produced a series of antigalactose (anti-Gal) hybridomas and characterized their heavy chain gene usage. Here we have quantified the affinity of these Abs for the alpha-Gal epitope and characterized their in vitro effects on endothelial cell activation and apoptosis. We report that anti-Gal mAbs derived from Gal(-/-) mice show a range of affinity for the alpha-Gal epitope, and that affinity was generally increased as the V(H) gene usage transitioned from germline sequences to sequences exhibiting somatic maturation. Despite an 85-fold range in affinity, all the anti-Gal mAbs examined induced alpha-Gal-specific endothelial cell activation, and after prolonged exposure induced endothelial cell apoptosis in a complement-independent manner. Only murine anti-Gal mAbs of the IgM or IgG3 subclass, but not IgG1, were effective at initiating complement-dependent cell lysis. Using a novel rat to mouse xenograft model, we examined the in vivo ability of these mAbs to induce xenograft rejection and characterized the rejection using histology and immunohistochemistry. Infusion of complement-fixing IgG3 mAbs resulted in either hyperacute rejection or acute vascular rejection of the xenograft. Surprisingly, infusion of an equal amount of a high affinity anti-Gal IgG1 mAb, that fixed complement poorly also induced a rapid xenograft rejection, which we have labeled very acute rejection. These studies emphasize the importance of in vivo assays, in addition to in vitro assays, in understanding the role of anti-Gal IgG-mediated tissue injury and xenograft rejection.     

The relationship between sequence and interaction divergence in proteins

There is currently a gap in knowledge between complexes of known three-dimensional structure and those known from other experimental methods such as affinity purifications or the two-hybrid system. This gap can sometimes be bridged by methods that extrapolate interaction information from one complex structure to homologues of the interacting proteins. To do this, it is important to know if and when proteins of the same type (e.g. family, superfamily or fold) interact in the same way. Here, we study interactions of known structure to address this question. We found all instances within the structural classification of proteins database of the same domain pairs interacting in different complexes, and then compared them with a simple measure (interaction RMSD). When plotted against sequence similarity we find that close homologues (30-40% or higher sequence identity) almost invariably interact the same way. Conversely, similarity only in fold (i.e. without additional evidence for a common ancestor) is only rarely associated with a similarity in interaction. The results suggest that there is a twilight zone of sequence similarity where it is not possible to say whether or not domains will interact similarly. We also discuss the rare instances of fold similarities interacting the same way, and those where obviously homologous proteins interact differently.     

Crystal structure of an archaeal class I aldolase and the evolution of (betaalpha)8 barrel proteins

Fructose-1,6-bisphosphate aldolase (FBPA) catalyzes the reversible cleavage of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate in the glycolytic pathway. FBPAs from archaeal organisms have recently been identified and characterized as a divergent family of proteins. Here, we report the first crystal structure of an archaeal FBPA at 1.9-A resolution. The structure of this 280-kDa protein complex was determined using single wavelength anomalous dispersion followed by 10-fold non-crystallographic symmetry averaging and refined to an R-factor of 14.9% (Rfree 17.9%). The protein forms a dimer of pentamers, consisting of subunits adopting the ubiquitous (betaalpha)8 barrel fold. Additionally, a crystal structure of the archaeal FBPA covalently bound to dihydroxyacetone phosphate was solved at 2.1-A resolution. Comparison of the active site residues with those of classical FBPAs, which share no significant sequence identity but display the same overall fold, reveals a common ancestry between these two families of FBPAs. Structural comparisons, furthermore, establish an evolutionary link to the triosephosphate isomerases, a superfamily hitherto considered independent from the superfamily of aldolases.     

Field trials of spinosad as a replacement for naled, DDVP, and malathion in methyl eugenol and cue-lure bucket traps to attract and kill male oriental fruit flies and melon flies (Diptera: Tephritidae) in Hawaii

Spinosad was evaluated in Hawaii as a replacement for organophosphate insecticides (naled, dichlorvos [DDVP], and malathion) in methyl eugenol and cue-lure bucket traps to attract and kill oriental fruit fly, Bactrocera dorsalis Hendel, and melon fly, B. cucurbitae Coquillett, respectively. In the first and second methyl eugenol trials with B. dorsalis, naled was in the highest rated group for all evaluation periods (at 5, 10, 15, and 20 wk). Spinosad was equal to naled at 5 and 10 wk during both trials 1 and 2, and compared favorably with malathion during trial 2. During the first cue-lure trial with B. cucurbitae, naled and malathion were in the top rated group at 5, 10, 15, and 20 wk. Spinosad was equal to naled at 5 wk. During the second cue-lure trial, spinosad and naled were both in the top rated group at 10, 15, and 20 wk. Use of male lure traps with methyl eugenol or cue-lure had no effect on attraction of females into test areas. Our results suggest that spinosad, although not as persistent as naled or malathion, is safer to handle and a more environmentally friendly substitute for organophosphate insecticides in methyl eugenol and cue-lure traps for use in B. dorsalis and B. cucurbitae areawide integrated pest management programs in Hawaii.     

Root responses and nitrogen acquisition by<Emphasis Type="Italic"> Artemisia tridentata</Emphasis> and<Emphasis Type="Italic"> Agropyron desertorum</Emphasis> following small summer rainfall events

Resources in the Great Basin of western North America often occur in pulses, and plant species must rapidly respond to temporary increases in water and nutrients during the growing season. A field study was conducted to evaluate below ground responses of Artemisia tridentata and Agropyron desertorum, common Great Basin shrub and grass species, respectively, to simulated 5-mm (typical summer rain) and 15-mm (large summer rain) summer rainfall events. The simulated rainfall was labeled with K(15)NO(3) so that timing of plant nitrogen uptake could be monitored. In addition, soil NH(4)(+) and NO(3)(-) concentrations and physiological uptake capacities for NO(3)(-) and NH(4)(+) were determined before and after the rainfall events. Root growth in the top 15 cm of soil was monitored using a minirhizotron system. Surprisingly, there was no difference in the amount of labeled N acquired in response to the two rainfall amounts by either species during the 7-day sample period. However, there were differences between species in the timing of labeled N uptake. The N label was detected in above ground tissue of Agropyron within 1 h of the simulated rainfall events, but not until 24 h after the rainfall in Artemisia. For both Agropyron and Artemisia, root uptake capacity was similarly affected by the 5-mm and 15-mm rainfall. There was, however, a greater increase in uptake capacity for NH(4)(+) than for NO(3)(-), and the 15-mm event resulted in a longer response. No root growth occurred in either species in response to either rainfall event during this 8-day period. The results of this study indicate that these species are capable of utilizing nitrogen pulses following even small summer rainfall events during the most stressful period of the summer and further emphasize the importance of small precipitation events in arid systems.     

Maintenance of the inner cell mass in human blastocysts from fragmented embryos

The degree of fragmentation during early cleavage is universally used as an indicator of embryo quality during human in vitro fertilization treatment. Extensive fragmentation has been associated with reduced blastocyst formation and implantation. We examined the relationship between early fragmentation and subsequent allocation of cells to the trophectoderm and inner cell mass in the human blastocyst. We retrospectively analyzed data from 363 monospermic human embryos that exhibited varying degrees of fragmentation on Day 2. Embryos were cultured from Day 2 to Day 6 in Earle balanced salt solution with 1 mM glucose and human serum albumin. Rates of development and blastocyst formation were measured. The number of cells in the trophectoderm and inner cell mass and the incidence of apoptosis were assessed following differential labeling with polynucleotide-specific fluorochromes. Increasing fragmentation resulted in reduced blastocyst formation and lower blastocyst cell numbers. For minimal and moderate levels of fragmentation, the reduction in cell numbers was confined largely to the trophectoderm and a steady number of inner cell mass cells was maintained. However, with extensive fragmentation of more than 25%, cell numbers in both lineages were reduced in the few embryos that formed blastocysts. Apoptotic nuclei were present in both the trophectoderm and inner cell mass, with the lowest incidence in blastocysts that had developed from embryos with minor (5-10%) fragmentation. Paradoxically, higher levels of apoptosis were seen in embryos of excellent morphology, suggesting a possible role in regulation of cell number.