ω-Helices in Proteins

A modification of the α-helix, termed the ω-helix, has four residues in one turn of a helix. We searched the ω-helix in proteins by the HELFIT program which determines the helical parameters—pitch, residues per turn, radius, and handedness—and p = rmsd/(N ? 1)^1/2 estimating helical regularity, where “rmsd” is the root mean square deviation from the best fit helix and “N” is helix length. A total of 1,496 regular α-helices 6–9 residues long with p ≤ 0.10 Å were identified from 866 protein chains. The statistical analysis provides a strong evidence that the frequency distribution of helices versus n indicates the bimodality of typical α-helix and ω-helix. Sixty-two right handed ω-helices identified (7.2% of proteins) show non-planarity of the peptide groups. There is amino acid preference of Asp and Cys. These observations and analyses insist that the ω-helices occur really in proteins.     

Age- and Sex-Associated Effects on Acute-Phase Proteins in G?ttingen Minipigs

Göttingen minipigs are a useful model for diseases having an inflammatory component, and the associated use of acute-phase proteins (APP) as biomarkers of inflammation warrants establishment of their reference ranges. The objective of this study was to establish reference values for selected APP in Göttingen minipigs and to investigate the effects of age, sex, and various stimuli on these ranges. Serum concentrations of C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin, pig major acute-phase protein (PMAP), albumin, and porcine α-1 acid glycoprotein (PAGP) were evaluated in 4 age groups (6, 16, 24 and 40–48 wk) of male and female Göttingen minipigs. In addition, minipigs were tested under 2 housing conditions, after acute LPS challenge, and after diet-induced obesity with and without mild diabetes. Changing the pigs to a new environment induced significant increases in CRP, PMAP, haptoglobin and PAGP and a decrease in albumin. An acute LPS stimulus increased CRP, PMAP, haptoglobin, and SAA; PAGP was unchanged and albumin decreased. Obese pigs with and without diabetes showed increases in CRP and PAGP, albumin decreased, and haptoglobin and SAA were unchanged. PMAP was increased only in obese pigs without diabetes. In conclusion, reference values for CRP, PMAP, haptoglobin, SAA, PAGP and albumin were established for male and female Göttingen minipigs of different ages. These APP were influenced by age and sex, underlining the importance of considering these factors when designing and interpreting studies including aspects of inflammation. In addition, an APP response was verified after both acute and chronic stimuli. Abbreviations: APP, acute-phase proteins; APR, acute-phase response; CRP, C-reactive protein; HFD, high-fat diet; HFD+D, high fat diet + diabetes; PAGP, porcine α1 acid glycoprotein; PMAP, pig major acute-phase protein; SAA, serum amyloid AInflammation is involved in a number of important and increasingly widespread human diseases, including inflammatory bowel diseases, cancers, infections, metabolic diseases like obesity and diabetes, and cardiovascular diseases like atherosclerosis.^{1,5,7,11,20,41} The systemic response to inflammation is the acute-phase response (APR) which, together with innate immune responses, prevents infection, clears pathogens, and contributes to inflammation resolution and the healing process. The APR has been extensively described in humans^10,22 and other mammals,^8,14,29,31 and in all cases, it is regulated by cytokines including IL6 and TNFα.^21,30 The APR is activated by many different stimuli, including trauma, infection, stress, neoplasia, and inflammatory stimuli, resulting in significant changes in the circulating concentrations of the so-called acute-phase proteins (APP). The APP are synthetized primarily by the liver and can be divided into positive and negative APP depending on whether their concentration in plasma increases (positive) or decreases (negative) in response to a stimulus.¹⁰ In addition, they can be divided into major and minor APP, depending on the magnitude of their concentration change after a given stimulus.²² Because the concentrations of the APP change in response to a given stimulus, their serum or plasma levels can be used diagnostically as biomarkers of disease severity and progression or to evaluate the effect of various interventions.^8,14,31 The APP show different kinetics after a stimulus, with C-reactive protein (CRP) and serum amyloid A (SAA) displaying rapid increases and normalization after the stimulus has been removed, whereas haptoglobin shows a later and more prolonged response.^10,31 The APR may be transient and revert to normal with recovery, or it can persist, as during chronic conditions.²¹ Importantly, APP and their kinetics differ somewhat between species.³¹To further elucidate the involvement of inflammation in human diseases, accurate animal models of inflammation, including species validated biomarkers of inflammation, are needed. Mouse models are commonly used in many research areas, but their response to several different inflammatory conditions is not comparable to that of humans, and therefore the predictive validity of these models may be limited.³⁹ Pigs are highly comparable to humans with respect to anatomy and physiology,⁴⁴ and their APR to various stimuli has been described.^14,23,26 In general, the APR and the resulting changes in APP seem to be very similar in pigs compared with humans, with CRP, haptoglobin, and SAA being major positive APP and albumin being a negative APP.¹⁴ In humans, α1-acid glycoprotein (AGP) is a positive APP but has been reported to either increase,¹⁷ remain unchanged^23,45 or to decrease¹² in pigs, depending on the stimulus investigated. The concentrations of some of the major APP characterized in domestic pigs show significant effects of age and sex.^32,34 In addition to age and sex effects, significant differences in APP between herds have been observed, most likely reflecting different pathogenic pressures in the different herds.³² Furthermore, some indications exist for possible interbreed differences in APP concentrations, although this possibility has not been investigated in detail.¹²Minipigs are especially relevant in biomedical research, given their smaller size and well-defined microbiology and genetics.⁴ Göttingen minipigs are a useful model for several conditions involving inflammation and the APR, including infection,² obesity,¹⁹ diabetes²⁴ and atherosclerosis,¹⁸ and different APP have already been used as biomarkers in some of these models.² Therefore, existing data suggest that APP commonly applied in human medicine could be relevant in Göttingen minipigs as well. However, the APR and reference values of APP, including the potential influence of age and sex indicated in other studies, have not been investigated systematically in this breed.^12,32,34The objective of the current study was to establish reference values of selected APP in normal Göttingen minipigs, including evaluation of the possible effects of age and sex. In addition, the effects of housing condition and acute and chronic inflammatory stimuli were assessed.     

Domain-Opening and Dynamic Coupling in the α-Subunit of Heterotrimeric G Proteins

Heterotrimeric G proteins are conformational switches that turn on intracellular signaling cascades in response to the activation of G-protein-coupled receptors. Receptor activation by extracellular stimuli promotes a cycle of GTP binding and hydrolysis on the G protein α-subunit (Gα). Important conformational transitions occurring during this cycle have been characterized from extensive crystallographic studies of Gα. However, the link between the observed conformations and the mechanisms involved in G-protein activation and effector interaction remain unclear. Here we describe a comprehensive principal component analysis of available Gα crystallographic structures supplemented with extensive unbiased conventional and accelerated molecular dynamics simulations that together characterize the response of Gα to GTP binding and hydrolysis. Our studies reveal details of activating conformational changes as well as the intrinsic flexibility of the α-helical domain that includes a large-scale 60° domain opening under nucleotide-free conditions. This result is consistent with the recently reported open crystal structure of Gs, the stimulatory G protein for adenylyl cyclase, in complex with the α2 adrenergic receptor. Sets of unique interactions potentially important for the conformational transition are also identified. Moreover simulations reveal nucleotide-dependent dynamical couplings of distal regions and residues potentially important for the allosteric link between functional sites.Heterotrimeric G proteins undergo cycles of GTP-dependent conformational rearrangements and alterations of their oligomeric αβγ form to convey receptor signals to downstream effectors that control diverse cellular processes ranging from movement to division and differentiation. Interaction with activated receptor promotes the exchange of GDP for GTP on the G protein α subunit (Gα) and its separation from its βγ subunit partners (Gβγ). Both isolated Gα and Gβγ then interact with downstream effectors. GTP hydrolysis deactivates Gα, which reassociates with Gβγ, becoming ready to restart the cycle. Each of these stages has been subjected to extensive crystallographic studies with high-resolution structures of Gα in complex with GDP, GTP analog, Gβγ, and, most recently, the G-protein-coupled receptors now available. These studies have provided extensive mechanistic insight. However, a number of important questions remain, including:

• How do the distinct conformations evident in the accumulated structures interconvert?
• How do disease-associated mutations affect the fidelity of these transitions?
• And, critically, how do distal functional sites responsible for nucleotide and protein partner binding allosterically coordinate their activities?

Here we describe a comprehensive analysis of the accumulated Gα crystallographic structures supplemented with extensive conventional (cMD) and accelerated molecular dynamics (aMD) simulations (1) that together map the structural and dynamical features of Gα in different nucleotide states. These enhanced sampling simulations reveal the spontaneous interconversion between GDP and GTP conformations and also characterize large-scale opening motions of the α-helical domain (HD) that were not accessible to previous simulation studies (2–5). Furthermore, the current simulations results reveal a distinctive pattern of collective motions that provide evidence for a nucleotide-dependent network of dynamic communication between the active site and the receptor and effector binding sites.Principal component analysis of 53 Gα experimental structures homologous to transducin (Gαt) reveals that the major variation in accumulated structures is the concerted association/disassociation of three nucleotide-binding site loops termed the switch regions (SI, SII, and SIII). An additional small-scale (<10°) rotation of the HD relative to the main catalytic Ras-like domain (RasD) is also apparent (see Fig. S1 in the Supporting Material). The distinct conformation of SI–SIII regions gives rise to nucleotide-associated segregation of GDP- and GTP-analog-bound experimental structures along the PC1-PC2 plane. Interestingly, both GDP- and GTP-bound structures display a skewed distribution along the PC1-PC2 plane that arises from HD rotation. In comparison, the distribution of the GTP-bound structures becomes more restricted and the skew decreases when the mapping is based on a principle component analysis that excludes the HD region (see Fig. S1).Recently, the HD region of Gαs (the α-subunit of the stimulatory G protein for adenyl cyclase) was shown to adopt a dramatically more open conformation in a crystal structure complex with the β2 adrenergic receptor (β2AR) (6). This clam-shell-like 127° opening in the absence of nucleotide and presence of receptor is consistent with electron microscopy (7) and double electron-electron resonance analysis (8). These results, together with recent hydrogen-deuterium exchange mass spectrometry data (9), indicate that there may be additional functional motions and inherent flexibility in the ensemble of native states beyond those apparent in the accumulated crystal structures of Gαt (9). To address this question, we performed multiple 100-ns aMD simulations of nucleotide-free Gαt. These simulations reveal a spontaneous large-scale opening and closing motion of larger magnitude (>60°) than those evident in the distribution of crystallographic structures (Fig. 1 A and see Fig. S2). In addition, the trajectory reveals two dominant modes of HD opening: an out-of-plane shifting (PC1 in Fig. S3) and an in-plane rotation (PC2 in Fig. S3). It is also notable that nucleotide-free aMD simulations sample both active (GTP-like) and inactive (GDP-like) structures (see Fig. S2) in an analogous manner to the spontaneous GDP to GTP interconversion sampled for Ras and Rho small G proteins with similar methods (10–12).Open in a separate windowFigure 1Nucleotide-associated differences in flexibility and dynamic coupling. (A) Mapping aMD simulation trajectories (blue points) onto the principal components obtained from analysis of Gα crystallographic GDP-bound (green) and GTP-analog bound (red) experimental structures. (Orange) Open β2AR-Gαs complex structure. (B) Results of dynamic coupling analysis mapped onto the average structure for each nucleotide state. (Spheres) Nodes for the nucleotide; the protein cartoon is colored by community structure. (C) Community network graph. (Circles) Communities, colored as in panel B. Radius of the circle indicates the number of residues in the community. Thickness of linking lines is determined by the maximum betweenness of the respective intercommunity edges (see the Supporting Material). (Red, blue, and green edges) Major topological difference between states.The low sequence identity between Gαt and Gαs (44.5%), as well as the absence of the receptor and Gβγ in the simulations, may explain the difference between the predicted ∼60° Gαt-HD rotation and that displayed in the β2AR-Gαs crystallographic structure (see Fig. S3). It is notable that, although the amplitude is much smaller, aMD simulations with bound nucleotide display similar dominant HD motions to those observed in the nucleotide-free simulations (see Fig. S4). This suggests that the interdomain flexibility of RasD and HD is likely an intrinsic feature of Gαt regardless of nucleotide state.The transition between distinct conformations (structural clusters; see Fig. S5) was observed to correspond to significant dynamical changes in side-chain contacts (see Fig. S6). Specifically, we found sequential contacts breaking during the HD in-plane rotation motion starting from the region between HD helix αD and RasD helix αG toward that between HD helix αE and RasD SIII and the P-loop. In comparison, for the out-of-plane shift, we found simultaneous breaking and formation of contacts in the region containing the loop between helices αB and αC, the N-terminus of αA, αE, and αF of HD; α1, SI, and the loop between strand β6 and helix α5 of RasD. Interactions highlighted in these regions as potentially important for the conformational transitions include D137::K276, S140::K273, S140::D227, Q143::R238, N145::E39, and D146::K266, the effect of which can be further evaluated by mutagenesis experiments and simulations.Dynamic network analysis methods developed by Sethi et al. (13) were used to examine whether the motions of one residue were correlated to the motions of another (distant) residue. In this approach, a weighted graph is constructed where each residue represents a node and the weight of the connection between nodes represents their respective correlation value. A clustering of edges is then used to define local communities of highly correlated residues that represent substructures that are highly intraconnected, but loosely interconnected. Applying this approach to multiple 40-ns cMD simulations initiated from GTP-, GDP-, and aMD-derived APO conformations revealed a consistent community composition as well as a distinct pattern of intercommunity connection between nucleotide states (Fig. 1, B and C).The dynamics of the RasD region can be decomposed into two main communities that stem from the nucleotide base and phosphate regions in GDP and GTP states: The first community is composed of residues from the P-loop, helix α1, strands β1–β3, and the phosphates of the nucleotide (orange in Fig. 1, B and C). The second community comprises residues from helix αG, strands β4–β6, and the nucleotide base region (tan in Fig. 1, B and C). This dynamic partitioning of the central β-sheet and central role of the nucleotide is consistent with the bilobal structure and dynamics previously reported for Ras (14). In the presence of GTP, the first community includes or is dynamically coupled to SI, SII, and SIII regions (see the orange node and the red edge in Fig. 1 C). Removal of the γ-phosphate of GTP disrupts this region, leading to decoupling of the switch regions from the nucleotide. Also evident for GDP states is an apparent tighter coupling of RasD and HD regions (blue edges in Fig. 1 C). We note that these findings are robust to the choice of initial simulation conditions and are observed in both cMD and aMD simulations (see Fig. S7 and Fig. S8). Nucleotide-free Gαt simulations display an altered dynamical network with respect to those of nucleotide bound states. In particular, RasD and HD regions lose connecting edges consistent with the large-scale opening of these domains (e.g., SIII-HD green edges in Fig. 1 C).A number of residues highlighted here as potentially important for mediating the coupling between prominent communities (see Table S1 in the Supporting Material) have been shown by previous mutagenesis studies to affect GDP release. For example, the double mutation A322S/R174M was found to significantly enhance the rate of GDP release (15). The current results indicate that these positions are involved in coupling the nucleotide and RasD. Also, mutations R144A and L232Q caused a faster basal GDP release rate in Gαi1 (16). The current analysis indicates that the equivalent positions in Gαt (S140 and M228) couple the RasD and HD, and suggests that their mutation could promote domain-domain motions. We also note the apparent coupling of α5 with the nucleotide base and Ploop-β1 with the phosphate regions of GDP. These direct connections of the receptor connecting N- and C-terminus to GDP are suggestive of potential routes for receptor-mediated GDP release. We expect further study of these sites and of receptor-bound dynamics to be informative in this regard.In conclusion, simulations suggest a flexible HD in Gαt similar to that found for Gαs. In particular, in the absence of nucleotide we observed the spontaneous large-scale opening and closing of HD relative to RasD, which was unseen in previous computational studies. Moreover, we found that the functional states of Gαt are associated with the distinct dynamical couplings of functional regions including SI–SIII, P-loop, α5, and the HD region. Finally, our results indicate that nucleotide may not directly induce large-scale conformational changes but, instead, act as a modulator of intrinsically accessible conformations and as a central participant in their associated dynamical couplings.     

Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins

G protein α subunits cycle between active and inactive conformations to regulate a multitude of intracellular signaling cascades. Important structural transitions occurring during this cycle have been characterized from extensive crystallographic studies. However, the link between observed conformations and the allosteric regulation of binding events at distal sites critical for signaling through G proteins remain unclear. Here we describe molecular dynamics simulations, bioinformatics analysis, and experimental mutagenesis that identifies residues involved in mediating the allosteric coupling of receptor, nucleotide, and helical domain interfaces of Gα_i. Most notably, we predict and characterize novel allosteric decoupling mutants, which display enhanced helical domain opening, increased rates of nucleotide exchange, and constitutive activity in the absence of receptor activation. Collectively, our results provide a framework for explaining how binding events and mutations can alter internal dynamic couplings critical for G protein function.     

DHHC5 Mediates β-Adrenergic Signaling in Cardiomyocytes by Targeting Gα Proteins

S-palmitoylation is a reversible posttranslational modification that plays an important role in regulating protein localization, trafficking, and stability. Recent studies have shown that some proteins undergo extremely rapid palmitoylation/depalmitoylation cycles after cellular stimulation supporting a direct signaling role for this posttranslational modification. Here, we investigated whether β-adrenergic stimulation of cardiomyocytes led to stimulus-dependent palmitoylation of downstream signaling proteins. We found that β-adrenergic stimulation led to rapidly increased Gαs and Gαi palmitoylation. The kinetics of palmitoylation was temporally consistent with the downstream production of cAMP and contractile responses. We identified the plasma membrane-localized palmitoyl acyltransferase DHHC5 as an important mediator of the stimulus-dependent palmitoylation in cardiomyocytes. Knockdown of DHHC5 showed that this enzyme is necessary for palmitoylation of Gαs, Gαi, and functional responses downstream of β-adrenergic stimulation. A palmitoylation assay with purified components revealed that Gαs and Gαi are direct substrates of DHHC5. Finally, we provided evidence that the C-terminal tail of DHHC5 can be palmitoylated in response to stimulation and such modification is important for its dynamic localization and function in the plasma membrane. Our results reveal that DHHC5 is a central regulator of signaling downstream of β-adrenergic receptors in cardiomyocytes.     

G Protein–Coupled Receptor-Type G Proteins Are Required for Light-Dependent Seedling Growth and Fertility in Arabidopsis

G protein–coupled receptor-type G proteins (GTGs) are highly conserved membrane proteins in plants, animals, and fungi that have eight to nine predicted transmembrane domains. They have been classified as G protein–coupled receptor-type G proteins that function as abscisic acid (ABA) receptors in Arabidopsis thaliana. We cloned Arabidopsis GTG1 and GTG2 and isolated new T-DNA insertion alleles of GTG1 and GTG2 in both Wassilewskija and Columbia backgrounds. These gtg1 gtg2 double mutants show defects in fertility, hypocotyl and root growth, and responses to light and sugars. Histological studies of shoot tissue reveal cellular distortions that are particularly evident in the epidermal layer. Stable expression of GTG1_pro:GTG1-GFP (for green fluorescent protein) in Arabidopsis and transient expression in tobacco (Nicotiana tabacum) indicate that GTG1 is localized primarily to Golgi bodies and to the endoplasmic reticulum. Microarray analysis comparing gene expression profiles in the wild type and double mutant revealed differences in expression of genes important for cell wall function, hormone response, and amino acid metabolism. The double mutants isolated here respond normally to ABA in seed germination assays, root growth inhibition, and gene expression analysis. These results are inconsistent with their proposed role as ABA receptors but demonstrate that GTGs are fundamentally important for plant growth and development.     

Genetic and Physical Interactions between Gα Subunits and Components of the Gβγ Dimer of Heterotrimeric G Proteins in Neurospora crassa

Heterotrimeric G proteins are critical regulators of growth and asexual and sexual development in the filamentous fungus Neurospora crassa. Three Gα subunits (GNA-1, GNA-2, and GNA-3), one Gβ subunit (GNB-1), and one Gγ subunit (GNG-1) have been functionally characterized, but genetic epistasis relationships between Gβ and Gα subunit genes have not been determined. Physical association between GNB-1 and FLAG-tagged GNG-1 has been previously demonstrated by coimmunoprecipitation, but knowledge of the Gα binding partners for the Gβγ dimer is currently lacking. In this study, the three N. crassa Gα subunits are analyzed for genetic epistasis with gnb-1 and for physical interaction with the Gβγ dimer. We created double mutants lacking one Gα gene and gnb-1 and introduced constitutively active, GTPase-deficient alleles for each Gα gene into the Δgnb-1 background. Genetic analysis revealed that gna-3 is epistatic to gnb-1 with regard to negative control of submerged conidiation. gnb-1 is epistatic to gna-2 and gna-3 for aerial hyphal height, while gnb-1 appears to act upstream of gna-1 and gna-2 during aerial conidiation. None of the activated Gα alleles restored female fertility to Δgnb-1 mutants, and the gna-3^Q208L allele inhibited formation of female reproductive structures, consistent with a need for Gα proteins to cycle through the inactive GDP-bound form for these processes. Coimmunoprecipitation experiments using extracts from the gng-1-FLAG strain demonstrated that the three Gα proteins interact with the Gβγ dimer. The finding that the Gβγ dimer interacts with all three Gα proteins is supported by epistasis between gnb-1 and gna-1, gna-2, and gna-3 for at least one function.     

De Novo Mutations in GNAO1, Encoding a Gαo Subunit of Heterotrimeric G Proteins,Cause Epileptic Encephalopathy

Heterotrimeric G proteins, composed of α, β, and γ subunits, can transduce a variety of signals from seven-transmembrane-type receptors to intracellular effectors. By whole-exome sequencing and subsequent mutation screening, we identified de novo heterozygous mutations in GNAO1, which encodes a Gα_o subunit of heterotrimeric G proteins, in four individuals with epileptic encephalopathy. Two of the affected individuals also showed involuntary movements. Somatic mosaicism (approximately 35% to 50% of cells, distributed across multiple cell types, harbored the mutation) was shown in one individual. By mapping the mutation onto three-dimensional models of the Gα subunit in three different complexed states, we found that the three mutants (c.521A>G [p.Asp174Gly], c.836T>A [p.Ile279Asn], and c.572_592del [p.Thr191_Phe197del]) are predicted to destabilize the Gα subunit fold. A fourth mutant (c.607G>A), in which the Gly203 residue located within the highly conserved switch II region is substituted to Arg, is predicted to impair GTP binding and/or activation of downstream effectors, although the p.Gly203Arg substitution might not interfere with Gα binding to G-protein-coupled receptors. Transient-expression experiments suggested that localization to the plasma membrane was variably impaired in the three putatively destabilized mutants. Electrophysiological analysis showed that Gα_o-mediated inhibition of calcium currents by norepinephrine tended to be lower in three of the four Gα_o mutants. These data suggest that aberrant Gα_o signaling can cause multiple neurodevelopmental phenotypes, including epileptic encephalopathy and involuntary movements.     

Do Unique Proteins Exist in Taste Buds?

Proteins in papillae on the bovine tongue were analyzed by semi-micro, polyacrylamide gel electrophoresis. All the proteins in the papillae with taste buds were observed to be common to proteins in the surrounding epithelium without taste buds. The protein band which was reported to form a weak complex with compounds called sweet by man was also found in all parts of the tongue epithelium. The receptor molecules for chemical stimuli may be distributed in all the cells of the tongue epithelium or the content of receptor molecules in taste bud papillae may be extremely low.     

π–π Interactions in Structural Stability: Role in RNA Binding Proteins

RNA binding proteins play significant roles in many bio-macromolecular systems. Aromatic amino acid residues are vital for several biological functions. In the present work, the influences of π–π interactions in RNA binding proteins are analyzed. There are a total of 3,396 π-residues in RNA binding proteins out of which 1,547, 1,241, and 608 are phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp), respectively. Among these 945, 634, and 356 Phe, Tyr, and Trp residues, respectively, are involved in π–π interactions. The observations indicate that majority of the aromatic residues in RNA binding proteins are involved in π–π interactions. Side chain–side chain π–π interactions are the predominant type of interactions in RNA binding proteins. These π–π interactions stabilize the core regions within RNA binding proteins. π–π interacting residues are evolutionary conserved. Residue-wise analysis indicates that π–π interacting residues have higher long-range contacts and hence they are important in the global conformational stability of these proteins.     

Double Suppression of the Gα Protein Activity by RGS Proteins

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FAMCS： Finding All Maximal Common Substructures in Proteins

Finding the common substructures shared by two proteins is considered as one of the central issues in computational biology because of its usefulness in understanding the structure-function relationship and application in drug and vaccine design. In this paper, we propose a novel algorithm called FAMCS （Finding All Maximal Common Substructures） for the common substructure identification problem. Our method works initially at the protein secondary structural element （SSE） level and starts with the identification of all structurally similar SSE pairs. These SSE pairs are then merged into sets using a modified Apriori algorithm, which will test the similarity of various sets of SSE pairs incrementally until all the maximal sets of SSE pairs that deemed to be similar are found. The maximal common substructures of the two proteins will be formed from these maximal sets. A refinement algorithm is also proposed to fine tune the alignment from the SSE level to the residue level. Comparison of FAMCS with other methods on various proteins shows that FAMCS can address all four requirements and infer interesting biological discoveries.     

Is There a Phylogenetic Signal in Prokaryote Proteins?

Using the sequence information from nine completely sequenced bacterial genomes, we extract 32 protein families that are thought to contain orthologous proteins from each genome. The alignments of these 32 families are used to construct a phylogeny with the neighbor-joining algorithm. This tree has several topological features that are different from the conventional phylogeny, yet it is highly reliable according to its bootstrap values. Upon closer study of the individual families used, it is clear that the strong phylogenetic signal comes from three families, at least two of which are good candidates for horizontal transfer. The tree from the remaining 29 families consists almost entirely of noise at the level of bacterial phylum divisions, indicating that, even with large amounts of data, it may not be possible to reconstruct the prokaryote phylogeny using standard sequence-based methods. Received: 22 November 1998 / Accepted: 17 February 1999     

Putative Calmodulin-Binding Domains in Aflatoxin Biosynthesis–Regulatory Proteins

The inhibition of aflatoxin production by trifluoperazine, an anticalmodulin (CaM) agent and the relevance of Ca²⁺/CaM-dependent phosphorylation and dephosphorylation during aflatoxin biosynthesis was previously reported. To identify proteins that may be regulated by CaM, an in silico analysis for putative CaM-binding domains (CaMBDs) in the aflatoxin-related proteins of Aspergillus parasiticus was performed using the CaM target database. Interestingly, the key regulators of aflatoxin biosynthesis such as AflR and AflJ contained predicted CaMBDs at their C-termini. Furthermore, potential phosphorylation sites for CaM-kinase II were present within these CaMBDs. In addition to other aflatoxin biosynthesis enzymes—such as Vbs, DmtA and OmtA, and the VeA protein (known to regulate the expression of AflJ and AflR)—also showed the presence of putative CaMBDs. Although the present report reaffirms earlier observations on CaM-mediated regulation of aflatoxin biosynthesis, it also opens new avenues for identifying the specific targets of CaM and elucidating the exact mechanism of initiation and regulation of aflatoxin biosynthesis.     

Tau Proteins and Tauopathies in Alzheimer’s Disease

Alzheimer’s disease (AD) is characterized by progressive memory loss and cognitive function deficits. There are two major pathological hallmarks that contribute to the pathogenesis of AD which are the presence of extracellular amyloid plaques composed of amyloid-β (Aβ) and intracellular neurofibrillary tangles composed of hyperphosphorylated tau. Despite extensive research that has been done on Aβ in the last two decades, therapies targeting Aβ were not very fruitful at treating AD as the efficacy of Aβ therapies observed in animal models is not reflected in human clinical trials. Hence, tau-directed therapies have received tremendous attention as the potential treatments for AD. Tauopathies are closely correlated with dementia and immunotherapy has been effective at reducing tau pathology and improving cognitive deficits in animal models. Thus, in this review article, we discussed the pathological mechanism of tau proteins, the key factors contributing to tauopathies, and therapeutic approaches for tauopathies in AD based on the recent progress in tau-based research.     

Synthesis of Bacteriophage φ29 Proteins in Bacillus subtilis

Seventeen bacteriophage phi29 proteins were detected in ultraviolet light-irradiated Bacillus subtilis by autoradiography of polyacrylamide slab gels. The appearance of phi29 proteins occurred either before or concomitantly with viral DNA replication. Viral proteins detected early in the infectious cycle consisted of nine polypeptides ranging from 5,200 daltons to 54,000 daltons. Two of the early proteins were identified as, respectively, the major capsid protein and the protein comprising the filaments which extend from the head of the virus. Late phi29 proteins were composed of eight polypeptides ranging from 14,000 daltons to 95,000 daltons. Only three late proteins were noncapsid proteins. Among the early proteins, six were synthesized at diminishing rates late in the infectious cycle. One of the early proteins (protein 12) lacked histidine, whereas two (proteins 10 and 15) lacked tryptophan. Among the 17 proteins detected, 10 were viral noncapsid proteins. The amount of viral genetic information required to code for the 17 proteins detected in these experiments (81% of the potential genetic information of phi29 DNA) compares favorably with the genetic information detected as mRNA in a previous report, 85% of the potential information on the phi29 chromosome.     

Characterization of Mamalian GS-α Proteins Expressed in Yeast

Abstract

The guanine nucleotide regulatory protein, G_s, mediates transmembrane signaling by coupling membrane receptors to the stimulation of adenylyl cyclase activity. The full length coding sequences for the Mp 42-45,000, short form (S), and M₁= 46-52,000, long form (L), of the a-subunits of rat G_s were placed in yeast expression vectors under the regulatory control of the copper-inducible CUP 1 promoter and transformed into Saccharomvces cerevisiae. In the presence of 100 pM CuSOq, the transformed yeast expressed Gs-a mRNAs and proteins. In reconstitution experiments, rat Gs-a(S and L), solubilized from yeast membranes with 1% cholate, conferred NaF-, (-)isoproterenol, and guanine nudeotidedependent sensitivity to adenylyl cyclase catalytic units in S49 lymphoma cyc- cell membranes, which are devoid of endogenous G_s-a. G_s-a(S) demonstrated twice the activity of Gs-a(L) in reconstitution assays of fluoride-stimulated adenylyl cyclase activity. Comparison of Gs-a(S) expressed in yeast with Gs purified from rabbit liver or human erythrocytes showed that the crude recombinant protein was fully competent in reconstituting NaF-stimulated adenylyl cyclase activity, but was only 2-5% as potent as puriiied G,. Addition of bovine brain py subunits during reconstitution enhanced all parameters of adenylyl cyclase activity for Gq-a(S and L) obtained from yeaa. In contrast, transducin py only enhanced agonist-stimulated adenylyl cyclase activity for Gs-a(S and L) following reconstitution. These results demonstrate that the expression of functional mammalian Gs-a subunits in yeast may be useful for their biochemical characterization.