Disentangling the Complexity of HGF Signaling by Combining Qualitative and Quantitative Modeling.

Signaling pathways are characterized by crosstalk, feedback and feedforward mechanisms giving rise to highly complex and cell-context specific signaling networks. Dissecting the underlying relations is crucial to predict the impact of targeted perturbations. However, a major challenge in identifying cell-context specific signaling networks is the enormous number of potentially possible interactions. Here, we report a novel hybrid mathematical modeling strategy to systematically unravel hepatocyte growth factor (HGF) stimulated phosphoinositide-3-kinase (PI3K) and mitogen activated protein kinase (MAPK) signaling, which critically contribute to liver regeneration. By combining time-resolved quantitative experimental data generated in primary mouse hepatocytes with interaction graph and ordinary differential equation modeling, we identify and experimentally validate a network structure that represents the experimental data best and indicates specific crosstalk mechanisms. Whereas the identified network is robust against single perturbations, combinatorial inhibition strategies are predicted that result in strong reduction of Akt and ERK activation. Thus, by capitalizing on the advantages of the two modeling approaches, we reduce the high combinatorial complexity and identify cell-context specific signaling networks.

Transmission dynamics of oral polio vaccine viruses and vaccine-derived polioviruses on networks.

One drawback of oral polio vaccine (OPV) is the potential reversion to more transmissible, virulent circulating vaccine-derived polioviruses (cVDPVs), which may cause outbreaks of paralytic poliomyelitis. Previous modeling studies of the transmission of cVDPVs assume an unrealistic homogeneous mixing of the population and/or ignore that OPV viruses and cVDPVs compete for susceptibles, which we show is a key to understanding the dynamics of the transmission of cVDPVs. We examined the transmission of OPV viruses and cVDPVs on heterogeneous, dynamic contact networks using differential equation-based and individual-based models. Despite the lower transmissibility, OPV viruses may outcompete more transmissible cVDPVs in the short run by spreading extensively before cVDPVs emerge. If viruses become endemic, however, cVDPVs eventually dominate and force OPV viruses to extinction. This study improves our understanding of the emergence of cVDPVs and helps develop more detailed models to plan a policy to control paralytic polio associated with the continued use of OPV in many countries.

Whole exome sequencing for a patient with Rubinstein-Taybi syndrome reveals de novo variants besides an overt CREBBP mutation.

Rubinstein-Taybi syndrome (RSTS) is a rare condition with a prevalence of 1 in 125,000-720,000 births and characterized by clinical features that include facial, dental, and limb dysmorphology and growth retardation. Most cases of RSTS occur sporadically and are caused by de novo mutations. Cytogenetic or molecular abnormalities are detected in only 55% of RSTS cases. Previous genetic studies have yielded inconsistent results due to the variety of methods used for genetic analysis. The purpose of this study was to use whole exome sequencing (WES) to evaluate the genetic causes of RSTS in a young girl presenting with an Autism phenotype. We used the Autism diagnostic observation schedule (ADOS) and Autism diagnostic interview revised (ADI-R) to confirm her diagnosis of Autism. In addition, various questionnaires were used to evaluate other psychiatric features. We used WES to analyze the DNA sequences of the patient and her parents and to search for de novo variants. The patient showed all the typical features of Autism, WES revealed a de novo frameshift mutation in CREBBP and de novo sequence variants in TNC and IGFALS genes. Mutations in the CREBBP gene have been extensively reported in RSTS patients, while potential missense mutations in TNC and IGFALS genes have not previously been associated with RSTS. The TNC and IGFALS genes are involved in central nervous system development and growth. It is possible for patients with RSTS to have additional de novo variants that could account for previously unexplained phenotypes.

Inhibition of melanogenesis by 5,7-dihydroxyflavone (chrysin) via blocking adenylyl cyclase activity.

Due to its multiple biological activities, 5,7-dihydroxyflavone (chrysin) in propolis has gained attention as potentially useful therapeutics for various diseases. However, the efficacy of chrysin for the use of dermatological health has not been fully explored. To clarify the action mechanism of the skin protecting property of chrysin, we firstly investigated the molecular docking property of chrysin on the mammalian adenylyl cyclase, which is the key enzyme of cAMP-induced melanogenesis. We also examined the involvement of chrysin in alpha-MSH and forskolin-induced cAMP signaling within a cell based assay. In addition, we inquired into the inhibitory effect of chrysin on melanogenesis and found that the pretreatment with chrysin inhibited the forskolin-induced melanin contents significantly without annihilating the cell viability. These results strongly suggest that chrysin directly inhibits the activity of adenylyl cyclase, downregulates forskolin-induced cAMP-production pathway, consequently inhibiting melanogenesis. Thus, chrysin may also be used as an effective inhibitor of hyperpigmentation.

Modulation of the conductance-voltage relationship of the BK Ca channel by mutations at the putative flexible interface between two RCK domains.

Calcium-dependent gating of the large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel is conferred by the large cytosolic carboxyl terminus containing two domains of the regulator of K(+) conductance (RCK) and the high-affinity Ca(2+)-binding site (the Ca(2+)-bowl). In our previous study, we located the putative second RCK domain (RCK2) and demonstrated that it interacts directly with RCK1 via a hydrophobic "assembly interface". In this study, we tested the structural model of the other interface, the "flexible interface", by strategically positioning charge pairs across the putative interface. Several charge mutations on RCK2 affected the voltage-dependent activation of the channel. In particular, the Gly-to-Asp substitution at position 803 profoundly affected channel activation by stabilizing the open conformation of the channel with minimal effects on its Ca(2+) affinity and voltage sensitivity. Various mutations at Gly-803 shifted the channel's conductance-voltage curve either left or right over a 145-mV range. Since this residue is predicted to be in the first loop of RCK2 these results strongly suggest that this loop plays a critical role in determining the intrinsic equilibrium constant for channel opening, and they support the hypothesis that this loop is part of an interface that mediates conformational coupling between RCK1 and RCK2.

An overview of cardiac systems biology.

The cardiac system has been a major target for intensive studies in the multi-scale modeling field for many years. Reproduction of the action potential and the ionic currents of single cardiomyocytes, as well as the construction of a whole organ model is well established. Still, there are major hurdles to overcome in creating a realistic and predictive functional cardiac model due to the lack of a profound understanding of the complex molecular interactions and their outcomes controlling both normal and pathological cardiophysiology. The recent advent of systems biology offers the conceptual and practical frameworks to tackle such biological complexities. This review provides an overview of major themes in the developing field of cardiac systems biology, summarizing some of the high-throughput experiments and strategies used to integrate the datasets, and various types of computational approaches used for developing useful quantitative models capable of predicting complex biological behavior.

Engineering of protease variants exhibiting altered substrate specificity.

By using an improved genetic screening system, variants of the HAV 3CP protease which exhibit altered P2 specificity were obtained. We randomly mutated the His145, Lys146, Lys147, and Leu155 residues that constitute the S2 pocket of 3CP and then isolated variants that preferred substrates with Gln over the original Thr at the P2 position using a yeast-based screening method. One of the isolated variants cleaved the Gln-containing peptide substrate more efficiently in vitro, proving the efficiency of our method in isolating engineered proteases with desired substrate selectivity.

Crystal structure and functional studies reveal that PAS factor from Vibrio vulnificus is a novel member of the saposin-fold family.

PAS factor is a novel putative bacterial secretion factor thought to induce secretion of periplasmic proteins. We solved the crystal structure of PAS factor from Vibrio vulnificus at 1.8A resolution and found it to be comprised of five alpha helices that form an antiparallel bundle with an up-and-down topology, and to adopt the saposin-fold characteristic of a family of proteins that bind to membranes and lipids. PAS factor lacks the disulfide bridge characteristic of mammalian saposin-fold proteins; in fact, it shows no sequence homology with mammalian proteins. Nevertheless, the molecular architectures are similar, and the shared propensity for membrane interaction suggests strongly that PAS factor is another member of the saposin-fold family. Analysis of the CD spectra showed that PAS factor binds to membranes directly, while measurement of calcein dye leakage showed that PAS factor interacts strongly with liposomes composed of anionic phospholipids, making them leaky, but binds very weakly with liposomes composed of zwitterionic phospholipids. Moreover, by analyzing tryptophan fluorescence emission from four single-tryptophan mutants (V10W, T22W, F35W, and L70W), we identified the putative phospholipid-binding site of PAS factor. The resultant membrane destabilization likely mediates secretion of periplasmic proteins required for the in vivo survival and pathogenesis of V.vulnificus.

HCNet: a database of heart and calcium functional network.

SUMMARY: The Heart and Calcium functional Network (HCNet) database is a collection of functional gene modules calculated from the microarray data compendium available from the GEO database. It is a specialized database designed to assist experimentalists for cardiac calcium signaling research by providing the pre-calculated gene clusters and their potential correlation network in heart. In the current release of HCNet, 57 functional modules from 786 target genes obtained by a bi-clustering analysis of 381 microarray datasets are available. Detailed information of the clusters such as expression profiles, network diagrams is provided in two categories, heart-specific genes and heart-specific genes along with calcium toolkit genes. Overrepresented gene ontological categories and transcription factors in each cluster are also provided to infer the biological implications of the detected functional modules. AVAILABILITY: HCNet is available at http://sbrg2.gist.ac.kr/hcnet.

Overexpression and purification of the RyR1 pore-forming region.

Ryanodine receptor 1 (RyR1) is a large homotetrameric calcium channel that plays a pivotal role in skeletal muscle contraction. Sequence comparison and mutagenesis studies indicate that the pore architecture of RyR1, including the last two transmembrane helices and the luminal loop linking them, is similar to that of the bacterial KcsA K(+) channel. Here, we describe the overexpression and purification of the C-terminal polyhistidine-tagged RyR1 pore-forming region. The nonionic detergent lauryldimethylamine oxide (LDAO) was selected for solubilization of the protein based on its ability to extract the protein from the membrane and to maintain it in a monodisperse state. The protein was then purified using nickel-affinity chromatography and gel filtration. Gel filtration analysis confirmed that the RyR1 fragment containing the pore-forming region (amino acids 4829-5037) is sufficient to form a tetramer.

Crystallization and preliminary X-ray crystallographic analysis of PAS factor from Vibrio vulnificus.

Plasmid Achromobacter secretion (PAS) factor is a putative secretion factor that induces the secretion of periplasmic proteins. PAS factor from Vibrio vulnificus was crystallized at 294 K by the hanging drop vapor-diffusion method. It was isolated as a monomer during the purification procedures. The native crystal belongs to the F222 space group with unit cell parameters a=56.1, b=74.4, c=80.0 A, a=b=g=90 degrees. The crystal was soaked in cryoprotectant containing 1 M NaBr for 1 h for MAD phasing. The diffraction limit of the Br-MAD data set was 1.9 A using synchrotron X-ray irradiation at beam line BL-18B at the Photon Factory, Japan.

Crystallization and preliminary X-ray crystallographic analysis of the GluR0 ligand-binding core from Nostoc punctiforme.

GluR0 from Nostoc punctiforme (NpGluR0) is a bacterial homologue of the ionotropic glutamate receptor. The ligand-binding core of NpGluR0 was crystallized at 294 K using the hanging-drop vapour-diffusion method. The L-glutamate-complexed crystal belongs to space group C222(1), with unit-cell parameters a = 78.0, b = 145.1, c = 132.1 A. The crystals contain three subunits in the asymmetric unit, with a VM value of 2.49 A3 Da(-1). The diffraction limit of the L-glutamate complex data set was 2.1 A using synchrotron X-ray radiation at beamline BL-4A of the Pohang Accelerator Laboratory (Pohang, Korea).

Crystal structure of a cyanobacterial phytochrome response regulator.

The two-component signal transduction pathway widespread in prokaryotes, fungi, molds, and some plants involves an elaborate phosphorelay cascade. Rcp1 is the phosphate receiver module in a two-component system controlling the light response of cyanobacteria Synechocystis sp. via cyanobacterial phytochrome Cph1, which recognizes Rcp1 and transfers its phosphoryl group to an aspartate residue in response to light. Here we describe the crystal structure of Rcp1 refined to a crystallographic R-factor of 18.8% at a resolution of 1.9 A. The structure reveals a tightly associated homodimer with monomers comprised of doubly wound five-stranded parallel beta-sheets forming a single-domain protein homologous with the N-terminal activator domain of other response regulators (e.g., chemotaxis protein CheY). The three-dimensional structure of Rcp1 appears consistent with the conserved activation mechanism of phosphate receiver proteins, although in this case, the C-terminal half of its regulatory domain, which undergoes structural changes upon phosphorylation, contributes to the dimerization interface. The involvement of the residues undergoing phosphorylation-induced conformational changes at the dimeric interface suggests that dimerization of Rcp1 may be regulated by phosphorylation, which could affect the interaction of Rcp1 with downstream target molecules.

Crystallization and preliminary X-ray crystallographic analysis of orotate phosphoribosyltransferase from Helicobacter pylori.

Orotic acid phosphoribosyltransferase (PyrE) (EC 2.4.2.10) is a key enzyme in de novo uridine monophosphate (UMP) biosynthesis. It catalyzes the reaction between orotic acid and 5-phosphoribosyl-1-pyrophosphate (PRPP) to yield orotidine monophosphate (OMP), which is transformed to uridine monophosphate by decarboxylation. H. pylori PyrE was crystallized at 294 +/- 1 K by the hanging drop vapor-diffusion method. The crystals belong to the space group P2(1)2(1)2(1) with unit-cell dimensions a = 95.8, b = 104.9, c = 281.1 A, alpha = beta = gamma = 90 degrees. A set of diffraction data was collected to 3.29 A resolution using synchrotron X-ray radiation.

Crystallization and preliminary X-ray crystallographic studies of phosphoglycerate kinase from Thermus caldophilus.

We report the purification and crystallization of phosphoglycerate kinase from Thermus caldophilus (Tca). The enzyme crystallizes in the P2(1)2(1)2(1) space group (cell dimensions a = 65.1, b = 71.3, c = 80.2 A), with one molecule in the asymmetric unit. A complete set of diffraction data was collected from an orthorhombic crystal up to 1.8 A resolution.

Crystallization and X-ray analysis of NH3-dependent NAD+ synthetase from Helicobacter pylori.

The ubiquitous NAD(+) synthetase catalyzes the key step in the biosynthesis of nicotinamide adenine dinucleotide. NH3-dependent NAD(+) synthetase from Helicobacter pylori was purified to homogeneity and crystallized using PEG 1500 as a precipitant. The crystal diffracted up to a resolution of 2.3+ and was found to belong to space group C2 with unit cell dimensions of a = 93.8, b = 48.3, c = 64.2 A and alpha = gamma = 90, beta = 110.0 degrees.

Crystallization and preliminary X-ray analysis of class II fructose-1,6-bisphosphate aldolase from Thermus caldophilus.

In this study, we have crystallized class II fructose-1,6-bisphosphate aldolase (FBA) from Thermus caldophilus (Tca). Purified Tca FBA is a tetrameric enzyme of 305 residues, which crystallizes in the space group P2(1)2(1)2(1) (cell dimensions a = 98.9, b = 113.1, c = 115.7 A), with four molecules in the asymmetric unit. A complete diffraction data set was obtained from orthorhombic crystals at resolution of 2.2 A.

An engineered viral protease exhibiting substrate specificity for a polyglutamine stretch prevents polyglutamine-induced neuronal cell death.

BACKGROUND: Polyglutamine (polyQ)-induced protein aggregation is the hallmark of a group of neurodegenerative diseases, including Huntington's disease. We hypothesized that a protease that could cleave polyQ stretches would intervene in the initial events leading to pathogenesis in these diseases. To prove this concept, we aimed to generate a protease possessing substrate specificity for polyQ stretches. METHODOLOGY/PRINCIPAL FINDINGS: Hepatitis A virus (HAV) 3C protease (3CP) was subjected to engineering using a yeast-based method known as the Genetic Assay for Site-specific Proteolysis (GASP). Analysis of the substrate specificity revealed that 3CP can cleave substrates containing glutamine at positions P5, P4, P3, P1, P2', or P3', but not substrates containing glutamine at the P2 or P1' positions. To accommodate glutamine at P2 and P1', key residues comprising the active sites of the S2 or S1' pockets were separately randomized and screened. The resulting sets of variants were combined by shuffling and further subjected to two rounds of randomization and screening using a substrate containing glutamines from positions P5 through P3'. One of the selected variants (Var26) reduced the expression level and aggregation of a huntingtin exon1-GFP fusion protein containing a pathogenic polyQ stretch (HttEx1(97Q)-GFP) in the neuroblastoma cell line SH-SY5Y. Var26 also prevented cell death and caspase 3 activation induced by HttEx1(97Q)-GFP. These protective effects of Var26 were proteolytic activity-dependent. CONCLUSIONS/SIGNIFICANCE: These data provide a proof-of-concept that proteolytic cleavage of polyQ stretches could be an effective modality for the treatment of polyQ diseases.