Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs

SNARE [soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor] proteins are essential for membrane fusion and are conserved from yeast to humans. Sequence alignments of the most conserved regions were mapped onto the recently solved crystal structure of the heterotrimeric synaptic fusion complex. The association of the four alpha-helices in the synaptic fusion complex structure produces highly conserved layers of interacting amino acid side chains in the center of the four-helix bundle. Mutations in these layers reduce complex stability and cause defects in membrane traffic even in distantly related SNAREs. When syntaxin-4 is modeled into the synaptic fusion complex as a replacement of syntaxin-1A, no major steric clashes arise and the most variable amino acids localize to the outer surface of the complex. We conclude that the main structural features of the neuronal complex are highly conserved during evolution. On the basis of these features we have reclassified SNARE proteins into Q-SNAREs and R-SNAREs, and we propose that fusion-competent SNARE complexes generally consist of four-helix bundles composed of three Q-SNAREs and one R-SNARE.     

Conserved features in papillomavirus and polyomavirus capsids

Capsids of papilloma and polyoma viruses (papovavirus family) are composed of 72 pentameric capsomeres arranged on a skewed icosahedral lattice (triangulation number of seven, T = 7). Cottontail rabbit papillomavirus (CRPV) was reported previously to be a T = 7laevo (left-handed) structure, whereas human wart virus, simian virus 40, and murine polyomavirus were shown to be T = 7dextro (right-handed). The CRPV structure determined by cryoelectron microscopy and image reconstruction was similar to previously determined structures of bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 1 (HPV-1). CRPV capsids were observed in closed (compact) and open (swollen) forms. Both forms have star-shaped capsomeres, as do BPV-1 and HPV-1, but the open CRPV capsids are approximately 2 nm larger in radius. The lattice hands of all papillomaviruses examined in this study were found to be T = 7dextro. In the region of maximum contact, papillomavirus capsomeres interact in a manner similar to that found in polyomaviruses. Although papilloma and polyoma viruses have differences in capsid size (approximately 60 versus approximately 50 nm), capsomere morphology (11 to 12 nm star-shaped versus 8 nm barrel-shaped), and intercapsomere interactions (slightly different contacts between capsomeres), papovavirus capsids have a conserved, 72-pentamer, T = 7dextro structure. These features are conserved despite significant differences in amino acid sequences of the major capsid proteins. The conserved features may be a consequence of stable contacts that occur within capsomeres and flexible links that form among capsomeres.     

Conserved features of TBE1 transposons in ciliated protozoa

BACKGROUND: Accessory function (AF) is one way antigen presenting cells generate sufficient secondary signals for optimal T-cell proliferation and IL-2 production. In general, alveolar macrophages (AM) are inferior accessory cells in comparison to monocytes whereas in sarcoidosis AF of AM is increased. METHODS: We compared the accessory index (AI) of AM and peripheral blood monocytes (PBM) of 41 patients with inactive sarcoidosis (SAR I, n = 12); active sarcoidosis with new or progressing symptoms (SAR II, n = 19), active sarcoidosis with spontaneous remission (SAR III, n = 10), tuberculosis (TB, n = 12), hypersensitivity pneumonitis (HP, n = 12), Wegener's disease (WD, n = 2), undefined alveolitis (UA, n = 8) and chronic obstructive pulmonary disease (COPD, n = 6) by employing the histoincompatibility-insensitive Jurkat cells as indicator cells. RESULTS: Compared with the controls (1.08 +/- 0.3) AMs of all groups but SAR I (AI: 0.96 +/- 0.42) exhibited significantly increased AIs (SAR II: 3.6 +/- 3.9; SAR III: 3.2 +/- 2.4; TB: 2.8 +/- 2.2; HP: 3 +/- 2; UA: 2.7 +/- 2.3; COPD: 3.1 +/- 2.2; p < 0.05 for all comparisons). Only in HP, AI of PBM was significantly increased compared with controls (3 +/- 1.5, 1.3 +/- 0.5, respectively; p < 0.001). Alveolar macrophages from patients with arcoidosis, TB, and HB express the costimulatory molecule CD80 on their surface and anti-CD80 antibodies inhibited the IL-2 release of Jurkat cells in this system to 59 +/- 27%. CONCLUSIONS: Our data demonstrate that AM from patients with various diseases have the capability to act as competent accessory cells and that the reported accessory function of these cells is at least in part mediated by the expression of CD80.     

Conserved features in the active site of nonhomologous serine proteases

BACKGROUND: Serine protease activity is critical for many biological processes and has arisen independently in a few different protein families. It is not clear, though, the degree to which these protease families share common biochemical and biophysical properties. We have used a computer program to study the properties that are shared by four serine protease active sites with no overall structural or sequence homology. The program systematically compares the region around the catalytic histidines from the four proteins with a set of noncatalytic histidines, used as controls. It reports the three-dimensional locations and level of statistical significance for those properties that distinguish the catalytic histidines from the noncatalytic ones. The method of analysis is general and can be applied easily to other active sites of interest. RESULTS: As expected, some of the reported properties correspond to previously known features of the serine protease active site, including the catalytic triad and the oxyanion hole. Novel properties are also found, including the spatial distribution of charged, polar, and hydrophobic groups arranged to stabilize the catalytic residues, and a relative abundance of some residues (Val, Tyr, Leu, and Gly) around the active site. CONCLUSIONS: Our findings show that in addition to some properties common to all the proteases examined, there are a set of preferred, but not required, properties that can be reliably observed only by aligning the sites and comparing them with carefully selected statistical controls.     

Specific features of the structural state in radiation-resistant structural materials

The factors of accelerating the recombination of vacancies and interstitial atoms in the structure of structural materials during irradiation are considered. The efficiencies of the effects of the distortion factors induced by substitutional and interstitial atoms and short-range ordering in a crystal lattice on the recombination of point defects are estimated and compared.     

Correlations between biological activity and structural properties for two short homologous sequences in thymosin beta4 and gelsolin

Gelsolin and thymosin beta4 appear to be two important actin-associated proteins involved in the regulation of actin polymerization. It has been widely demonstrated that thymosin is the major cellular actin-sequestering factor shifting the polymerization equilibrium of actin towards a monomeric state. At the same time gelsolin, a Ca2+ and inositol phosphate sensitive protein, regulates actin filament length. The interactions of these two proteins with actin are rather complex and require the participation of several complementary peptide sequences. We have identified a common motif, (I, V)EKFD, in the two proteins in the functional sequences so far examined. Gelsolin- and thymosin beta4-related peptides including the common motif were synthesized and their structural and functional properties studied. These two sequences exert a major inhibitory effect on salt-induced actin polymerization. We used circular dichroism and Fourier-transform infrared spectroscopy to show that the two synthetic peptides present some secondary structure in solution. As far as the peptide derived from the thymosin sequence was concerned, alpha-helical structure was induced by trifluoroethanol as observed with the full-length molecule. These experiments underscore the importance of the conformational state of peptide fragments in their biological activities. ELISA and fluorescence measurements have been used to identify the binding regions of these fragments to a C-terminal region (subdomain 1) of the actin sequence. Our results also emphasize the relationship between the propensity of small sequences to form secondary structures and their propensity for biological activity as related to actin interaction and inhibition of actin polymerization.     

Making and structural features of Ti-N-B and Ti-N-C nanocomposites

The paper examines the high-pressure sintering of nanograined ceramic polycrystals based on TiN-TiB₂ and TiC_0.5N_0.5 refractory compounds. Using the optimum pressure (up to 4 GPa) allows keeping the initial nanostate (TiN-TiB₂ and TiC_0.5N_0.5) and obtaining high-density ceramics with enhanced mechanical properties. An x-ray structural analysis is used to examine how the TiN-TiB₂ and TiC_0.5N_0,5 crystalline structure evolves during temperature-pressure treatment, which produces new ceramic materials. Based on the properties of the polycrystalline materials obtained, the temperature-time mode for the consolidation of initial nanopowders is determined to ensure favorable parameters of sintered nanograined ceramics. __________ Translated from Poroshkovaya Metallurgiya, Vol. 47, No. 1–2 (459), pp. 62–72, 2008.     

Physicochemical and structural features of mono- and polycrystalline manganese-zinc ferrites

Studies carried out using various methods — x-ray structural and spectral, electromagnetic, gravimetric, nuclear -resonance, and nuclear magnetic resonance — revealed that the crystal-chemical structure of mono-and polycrystalline manganese — zinc ferrites used in video tape recorders and color television sets possess defects, with various degrees of localization (point, plane, and cluster), existing simultaneously in both the cation and anion sublattices. Differences in the structure of mono- and polycrystalline specimens were observed, but the specimens were similar with respect to the nature of the clustered defects. A relationship between the defect structure and specified electromagnetic properties of the ferrites was established. It is concluded that more information is needed concerning the actual crystal-chemical structure of ferrites containing defects of different types and dimensions (micro, meso, macro).VNII Reaktivélektron, Donetsk. Translated from Poroshkovaya Metallurgiya, No. 5–6, pp. 89–93, May–June, 1994.     

Conserved sequence and structural motifs contribute to the DNA binding and cleavage activities of a geminivirus replication protein

Tomato golden mosaic virus (TGMV), a member of the geminivirus family, has a single-stranded DNA genome that replicates through a rolling circle mechanism in nuclei of infected plant cells. TGMV encodes one essential replication protein, AL1, and recruits the rest of the DNA replication apparatus from its host. AL1 is a multifunctional protein that binds double-stranded DNA, catalyzes cleavage and ligation of single-stranded DNA, and forms oligomers. Earlier experiments showed that the region of TGMV AL1 necessary for DNA binding maps to the N-terminal 181 amino acids of the protein and overlaps the DNA cleavage (amino acids 1-120) and oligomerization (amino acids 134-181) domains. In this study, we generated a series of site-directed mutations in conserved sequence and structural motifs in the overlapping DNA binding and cleavage domains and analyzed their impact on AL1 function in vivo and in vitro. Only two of the fifteen mutant proteins were capable of supporting viral DNA synthesis in tobacco protoplasts. In vitro experiments demonstrated that a pair of predicted alpha-helices with highly conserved charged residues are essential for DNA binding and cleavage. Three sequence motifs conserved among geminivirus AL1 proteins and initiator proteins from other rolling circle systems are also required for both activities. We used truncated AL1 proteins fused to a heterologous dimerization domain to show that the DNA binding domain is located between amino acids 1 and 130 and that binding is dependent on protein dimerization. In contrast, AL1 monomers were sufficient for DNA cleavage and ligation. Together, these results established that the conserved motifs in the AL1 N terminus contribute to DNA binding and cleavage with both activities displaying nearly identical amino acid requirements. However, DNA binding was readily distinguished from cleavage and ligation by its dependence on AL1/AL1 interactions.     

Conserved structural regions involved in the catalytic mechanism of Escherichia coli K-12 WaaO (RfaI)

Escherichia coli K-12 WaaO (formerly known as RfaI) is a nonprocessive alpha-1,3 glucosyltransferase, involved in the synthesis of the R core of lipopolysaccharide. By comparing the amino acid sequence of WaaO with those of 11 homologous alpha-glycosyltransferases, four strictly conserved regions, I, II, III, and IV, were identified. Since functionally related transferases are predicted to have a similar architecture in the catalytic sites, it is assumed that these four regions are directly involved in the formation of alpha-glycosidic linkage from alpha-linked nucleotide diphospho-sugar donor. Hydrophobic cluster analysis revealed a conserved domain at the N termini of these alpha-glycosyltransferases. This domain was similar to that previously reported for beta-glycosyltransferases. Thus, this domain is likely to be involved in the formation of beta-glycosidic linkage between the donor sugar and the enzyme at the first step of the reaction. Site-directed mutagenesis analysis of E. coli K-12 WaaO revealed four critical amino acid residues.     

Peptidyl-transferase ribozymes: trans reactions, structural characterization and ribosomal RNA-like features

BACKGROUND: One of the most significant questions in understanding the origin of life concerns the order of appearance of DNA, RNA and protein during early biological evolution. If an 'RNA world' was a precursor to extant life, RNA must be able not only to catalyze RNA replication but also to direct peptide synthesis. Iterative RNA selection previously identified catalytic RNAs (ribozymes) that form amide bonds between RNA and an amino acid or between two amino acids. RESULTS: We characterized peptidyl-transferase reactions catalyzed by two different families of ribozymes that use substrates that mimic A site and P site tRNAs. The family II ribozyme secondary structure was modeled using chemical modification, enzymatic digestion and mutational analysis. Two regions resemble the peptidyl-transferase region of 23S ribosomal RNA in sequence and structural context; these regions are important for peptide-bond formation. A shortened form of this ribozyme was engineered to catalyze intermolecular ('trans') peptide-bond formation, with the two amino-acid substrates binding through an attached AMP or oligonucleotide moiety. CONCLUSIONS: An in vitro-selected ribozyme can catalyze the same type of peptide-bond formation as a ribosome; the ribozyme resembles the ribosome because a very specific RNA structure is required for substrate binding and catalysis, and both amino acids are attached to nucleotides. It is intriguing that, although there are many different possible peptidyl-transferase ribozymes, the sequence and secondary structure of one is strikingly similar to the 'helical wheel' portion of 23S rRNA implicated in ribosomal peptidyl-transferase activity.     

Some chemical and structural features of the conidial wall of Trichoderma viride

Cell wall of spores of Trichoderma viride contains polymers similar to those of mycelial cell wall, such as beta-(1 leads to 3), beta-(1 leads to 6) glucans and protein, but chitin, always present in the mycelium, cannot be found in spores. Melanin, which in other fungi appears associated with chitin, replaces this polymer in the spore wall of T. viride and is located in the outermost layer. Attempts to characterize the pigment of the spore wall indicate that it is a non-indolic melanin-like polyphenol.     

Subfamily of olfactory receptors characterized by unique structural features and expression patterns

The complex chemospecificity of the olfactory system is probably due to the large family of short-looped, heptahelical receptor proteins expressed in neurons widely distributed throughout one of the several zones within the nasal neuroepithelium. In this study, a subfamily of olfactory receptors has been identified that is characterized by distinct structural features as well as a unique expression pattern. Members of this receptor family are found in mammals, such as rodents and opossum, but not in lower vertebrates. All identified subtypes comprise an extended third extracellular loop that exhibits amphiphilic properties and contains numerous charged amino acids in conserved positions. Olfactory sensory neurons expressing these receptor types are segregated in small clusters on the tip of central turbinates, thus representing a novel pattern of expression for olfactory receptors. In mouse, genes encoding the new subfamily of receptors were found to be harbored within a small contiguous segment of genomic DNA. Based on species specificity as well as the unique structural properties and expression pattern, it is conceivable that the novel receptor subfamily may serve a special function in the olfactory system of mammals.     

Expression of a structural domain of the beta 2 subunit essential for alpha M beta 2 ligand recognition

The beta2 leukocyte integrins comprise a group of closely related adhesion receptors that mediate critical events during normal and inflammatory immune responses. Central to the understanding of beta2 integrin function is the basis of ligand recognition. Results from our laboratory and others indicate the presence of multiple ligand contact points in both the alpha and beta subunit. As an approach to identify and characterize regulatory domains of the beta2 subunit, we have generated two different subdomains of the beta2 subunit for expression on the surface of mammalian cells through a phosphatidyl-inositol glycan anchor. The first subdomain contains the putative beta2 MIDAS motif implicated in ligand binding [beta2(LB)], whereas the second beta2 subdomain contains the cysteine-rich region [beta2(CR)]. Cells expressing alphaM and beta2 constructs singly or cotransfected transiently in COS-7 cells were tested for the ability to bind to immobilized iC3b. Cells bearing the recombinant alphaMbeta2(LB) were capable of adhering to iC3b in a manner similar to that observed with the complete alphaMbeta2 heterodimer. In contrast, cells expressing alphaMbeta2(CR) failed to adhere to immobilized iC3b. Moreover, cells bearing singly transfected alpha or beta chains alone failed to adhere to immobilized iC3b. These results indicate that along with alphaM, the beta2(LB) subdomain contains the sufficient components within the beta2 subunit essential for ligand recognition. These findings support the hypothesis that the beta2 subunit cooperates with site(s) within the alphaM subunit in a receptor/cation/ligand complex resulting in high-affinity ligand interaction.     

Responses of auditory-cortex neurons to structural features of natural sounds

Sound-processing strategies that use the highly non-random structure of natural sounds may confer evolutionary advantage to many species. Auditory processing of natural sounds has been studied almost exclusively in the context of species-specific vocalizations, although these form only a small part of the acoustic biotope. To study the relationships between properties of natural soundscapes and neuronal processing mechanisms in the auditory system, we analysed sound from a range of different environments. Here we show that for many non-animal sounds and background mixtures of animal sounds, energy in different frequency bands is coherently modulated. Co-modulation of different frequency bands in background noise facilitates the detection of tones in noise by humans, a phenomenon known as co-modulation masking release (CMR). We show that co-modulation also improves the ability of auditory-cortex neurons to detect tones in noise, and we propose that this property of auditory neurons may underlie behavioural CMR. This correspondence may represent an adaptation of the auditory system for the use of an attribute of natural sounds to facilitate real-world processing tasks.     

Prion protein structural features indicate possible relations to signal peptidases

We describe the treatment of 2 patients with extensive midfacial defects by using a customized composite flap. Autogenous bone was transferred from the iliac crest, revascularized by the rectus muscle, and then transferred as a complete unit by microvascular technique to provide a vascularized bone graft to this area of difficult reconstruction. The bone was shaped and customized to fit the osseous defect. Although the rectus muscle was relatively bulky at the time of harvest, its hearty blood supply permitted trimming and contouring to fit the gap. This technique was based on the principles proposed previously by other authors regarding the vascularization of a free bone graft and its transfer as a composite flap. This technique requires more research and refinement, however this clinical demonstration shows the feasibility of revascularizing a composite flap and the extent to which this technique can be performed.     

Comparison between beta 3 and beta 2 adrenoceptor agonists as inhibitors of gastric acid secretion

In order to investigate the role of beta 3 adrenoceptors in the regulation of gastric acid secretion we studied the effects of compound SR58611A (a selective agonist for atypical beta adrenoceptors), alone or in combination with beta-adrenoceptor antagonists, in the gastric fistula of a conscious cat. The effects of SR58611A were compared with those of clenbuterol, a selective agonist for beta 2 adrenoceptors. Intravenous infusion of SR58611A (0.3-3 mumol/kg/h) caused a dose-dependent, but partial, inhibition of the acid secretory response to 2-deoxy-D-glucose 100 mg/kg i.v., maximum effect not exceeding 40%. Clenbuterol (0.03-0.1 mumol/kg/h) caused a similar effect (maximum inhibition about 50%) at doses approximately 30 times lower. The acid secretion induced by the histamine H2-receptor agonist dimaprit (1 mumol/kg/h) was minimally affected by both beta adrenoceptor agonists. The inhibitory effect of SR58611A (3 mumol/kg/h) on 2-deoxy-D-glucose-induced acid secretion was not modified by pretreatment with the non-selective beta 1- and beta 2-adrenoceptor blocker propranolol, administered at doses (1.5 mumol/kg iv) that completely blocked the inhibitory effect of clenbuterol (0.1 mumol/kg/h). In contrast, bupranolol (10 mumol/kg i.v.) (a drug endowed with beta 3 antagonistic properties) prevented the inhibitory effects of both SR58611A and clenbuterol. The present data provide functional evidence that, besides beta 2-, also beta 3-adrenoceptors can have negative effects on gastric acid secretion, particularly when it is stimulated by indirect stimuli, like 2-deoxy-D-glucose. This gastric antisecretory activity may represent an additional mechanism for the physio-pharmacological control of gastric acid secretion.