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The SnRK (sucrose non-fermenting-1 related protein kinase) families respond to salt tolerance, nutritional stress, disease stress, and carbon metabolic regulation. In this study, the SnRK2.8 gene was cloned from Arabidopsis thaliana and nominated AtSnRK2.8. Bioinformatics characterization of AtSnRK2.8 showed that it contained an ATP binding site and a serine/threonine protein kinase binding site at the N-terminal catalytic domain. In addition, the AtSnRK2.8 protein contained special domain I at the C-terminal catalytic domain. Real-time polymerase chain reaction (PCR) results showed that the AtSnRK2.8 gene was expressed in all tested tissues and was highly expressed in seeds. Our analyses proved that the AtSnRK2.8 gene expression was upregulated by salt and drought stresses. The recombinant AtSnRK2.8 protein was expressed using a pET-28a system and purified with HisTrap HP affinity columns. The recombinant AtSnRK2.8 protein has the properties of a protein kinase. The results of overexpressing AtSnRK2.8 in Nanlin895 poplar showed that the transgenic poplar plants had a better ability to resist drought and salt stress compared to the wild type, and adapted well to drought and a high-salt environment.

Allium mongolicum Regel (A. mongolicum) is a perennial and xerophytic Liliaceous allium plant in high altitude desert steppe and desert areas. Feeding A. mongolicum greatly reduced unpleasant mutton flavor and improves meat quality of sheep. We analyzed epigenetic regulatory mechanisms of water extracts of A. mongolicum (WEA) on sheep muscle and adipose using RNA-Seq and whole-genome Bisulfite sequencing. Feeding WEA reduced differentially expressed genes and long non-coding RNAs (lncRNAs) between two tissues but increased differentially methylation regions (DMRs). LncRNA and DMR targets were both involved in ATP binding, ubiquitin, protein kinase binding, regulation of cell proliferation, and related signaling pathways, but not unsaturated fatty acids metabolism. Besides, tissue specific targets were involved in distinct functional annotations, e.g., Golgi membrane and endoplasmic reticulum for muscle lncRNA, oxidative phosphorylation metabolism for adipose lncRNA, dsRNA binding for muscle DMRs. Epigenetic regulatory networks were also discovered to discovered essential co-regulated modules, e.g., co-regulated insulin secretion module (PDPK1, ATP1A2, CACNA1S and CAMK2D) in adipose. The results indicated that WEA induced distinct epigenetic regulation on muscle and adipose to diminish transcriptome differences between tissues, which highlights biological functions of A. mongolicum, tissue similarity and specificity, as well as regulatory mechanism of mutton odor.

Background Helicobacter pylori (H. pylori) infection has become an international public health problem, and antibiotic-based triple or quadruple therapy is currently the mainstay of treatment. However, the effectiveness of these therapies decreases due to resistance to multiple commonly used antibiotics. Sanguisorba officinalis L. (S. officinalis), a traditional Chinese medicine clinically used for hemostasis and treatment of diarrhea, has various pharmacological activities. In this study, in vitro antimicrobial activity was used for the preliminary evaluation of S. officinalis against H. pylori. And a pharmacology analysis approach was also utilized to elucidate its underlying mechanisms against H. pylori infection. Methods Micro-broth dilution method, agar dilution method, checkerboard assay, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) were used for the assessment of anti-bacterial activity. Active ingredients screening, GO analysis, KEGG analysis, construction of PPI network, molecular docking, and RT-qPCR were used to elucidate the underlying pharmacological mechanisms of S. officinalis against H. pylori infection. Results The minimum inhibitory concentration (MIC) values of S. officinalis against multiple H. pylori strains including clinically isolated multi-drug resistant (MDR) strains were ranging from 160 to 320 µg/ml. These results showed that S. officinalis had additive interaction with four commonly used antibiotics and could exert antibacterial effect by changing the morphology of bacteria without developing drug resistance. Through network pharmacology analysis, 8 active ingredients in S. officinalis were screened out for subsequent studies. Among 222 putative targets of S. officinalis, 49 targets were identified as potential targets for treatment of H. pylori infection. And these 49 targets were significantly enriched in GO processes such as protein kinase B signaling, protein kinase activity, protein kinase binding, and KEGG pathways such as Pathways in cancer, MicroRNAs in cancer, and TNF signaling pathway. Protein-protein interaction analysis yielded 5 core targets (AKT1, VEGFA, EGFR, SRC, CCND1), which were validated by molecular docking and RT-qPCR. Conclusions Overall, this study confirmed the in vitro inhibitory activity of S. officinalis against H. pylori and explored the possible pharmacological mechanisms, laying the foundation for further research and clinical application.

MicroRNAs (miRNAs) represent RNA species found in serum. Many miRNAs were observed that were related to osteoporosis and osteopenia. However, expression and function analysis of miRNAs in postmenopausal osteoporosis (PMOP) remain unaddressed. We first compared the miRNA expression of blood samples in postmenopausal women with osteopenia or with osteoporosis via analysis of GSE64433. Bioinformatics analyses were conducted to get the key miRNAs and their functions and pathways. 331 miRNAs were being identified as differentially expressed miRNAs. Among these, 122 miRNA (36.86%) were up-regulated, and the remaining 209 miRNAs (63.14%) were down-regulated. 105 genes were predicted as the targets of these miRNAs. GO enrichment analysis results showed that the miRNAs mainly enriched in DNA binding, ATP binding, gene expression, regulation of the apoptotic process, chromatin binding, and protein kinase binding. KEGG enrichment analysis results demonstrated that the miRNAs mainly enriched in the TGF beta signaling pathway, wnt signaling pathway, JAK-STAT signaling pathway, and androgen receptor signaling pathway. This study identified the abundant differentially expressed miRNAs in the blood samples of postmenopausal women with osteopenia or with osteoporosis. This study may contribute to getting new diagnostic and therapeutic strategies for PMOP.

Hongyi Liao,1,* Xianbiao Xie,2,* Yuanyuan Xu,3 Gang Huang2 1Department of Orthopedic Surgery, The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen, People’s Republic of China; 2Department of Orthopedic Oncology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, People’s Republic of China; 3Department of Pediatrics, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, People’s Republic of China *These authors contributed equally to this work Background: The aim of this study was to identify the driver genes associated with chemotherapy resistance of Ewing’s sarcoma and potential targets for Ewing’s sarcoma treatment.Methods: Two mRNA microarray datasets, GSE12102 and GSE17679, were downloaded from the Gene Expression Omnibus database, which contain 94 human Ewing’s sarcoma samples, including 65 from those who experienced a relapse and 29 from those with no evidence of disease. The differen­tially expressed genes (DEGs) were identified using LIMMA package R. Subsequently, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed for DEGs using Database for Annotation, Visualization and Integrated Analysis. The protein–protein interaction network was constructed using Cytoscape software, and module analysis was performed using Molecular Complex Detection.Results: A total of 206 upregulated DEGs and 141 downregulated DEGs were identified. Upregulated DEGs were primarily enriched in DNA replication, nucleoplasm and protein kinase binding for biological processes, cellular component and molecular functions, respectively. Downregulated DEGs were predominantly involved in receptor clustering, membrane raft, and ligand-dependent nuclear receptor binding. The protein–protein interaction network of DEGs consisted of 150 nodes and 304 interactions. Thirteen hub genes were identified, and biological analysis revealed that these genes were primarily enriched in cell division, cell cycle, and mitosis. Furthermore, based on closeness centrality, betweenness centrality, and degree centrality, the three most significant genes were identified as GAPDH, AURKA, and EHMT2. Furthermore, the significant network module was composed of nine genes. These genes were primarily enriched in mitotic nuclear division, mitotic chromosome condensation, and nucleoplasm.Conclusion: These hub genes, especially GAPDH, AURKA, and EHMT2, may be closely associated with the progression of Ewing’s sarcoma chemotherapy resistance, and further experiments are needed for confirmation. Keywords: Ewing’s sarcoma, chemotherapy resistance, differentially expressed gene, enrichment analysis, network

Mitogen-activated protein kinase binding protein 1 (MAPKBP1) is a key transcription factor in the NF-κB signalling pathway. In this study, associations between MAPKBP1 expression and molecular and clinical characteristics were evaluated by several microarray datasets. We found that MAPKBP1 was over-expressed in cytogenetically normal AML (CN-AML) patients compared to normal bone marrow. High MAPKBP1 expression (MAPKBP1high) was associated with significantly shorter event-free survival (EFS; P = 0.0004) and overall survival (OS; P = 0.0006) than low MAPKBP1 expression (MAPKBP1low) in a cohort of 157 CN-AML patients. In multivariable analyses, MAPKBP1high remained associated with shorter EFS (P = 0.003) and OS (P = 0.01). Validation in an independent cohort of 162 CN-AML patients further confirmed the prognostic value of MAPKBP1 (OS, P = 0.00172). Gene-expression profiling revealed that some important oncogenes, including MYCN, MYB, CDK6 and CCND2, etc, were up-regulated, while cell signalling pathways leading to apoptosis, antigen processing, and natural killer cell-mediated cytotoxicity were down-regulated in MAPKBP1high patients with CN-AML. MicroRNA expression profiling revealed thatsome oncogenic microRNAsincluding miR-155 and miR-126 were up-regulated, whilst anti-oncogenic microRNAsincluding miR-148a and miR-193a were down-regulated in MAPKBP1high patients with CN-AML, which may underlie the pathological processes in this malignancy. Taken together, these findings suggest MAPKBP1highis a novel, unfavourably prognostic biomarker for CN-AML risk-stratification.

Long-term consumption of sodium arsenite contaminated water can cause endemic arsenic disease. The proteome profile changes of liver fibrosis after exposure to arsenite containing water remain unclear. In this study, Sprague-Dawley (SD) male rats were treated with sodium arsenite (iAs3+), using a daily dose of 1.36 mg/kg body weight (medium dose group, M), 2.73 mg/kg body weight (high dose group, H) or deionized water (control group, C). Isobaric tags for relative and absolute quantitation (iTRAQ) were used to identify the different abundant proteins (DAPs) after arsenic-induced liver fibrosis. A total of 2987 high-quality proteins were detected (95% confident peptides ≥ 2), 608 of which were differentially expressed (fold change > 2 and p

Deregulation of protein kinases is associated with numerous diseases, making them important targets for drug discovery. The majority of drugs target the catalytic site of these proteins, but due to the high level of similarity within the ATP binding sites of protein kinases, it is often difficult to achieve the required pharmacological selectivity. In this study, we describe the identification and subsequent analysis of water patterns in the ATP binding sites of 171 protein kinase structures, comprising 19 different kinases from various branches of the kinome, and demonstrate that structurally similar binding sites often have significantly different water patterns. We show that the observed variations in water patterns of different, but structurally similar kinases can be exploited in the structure-based design of potent and selective kinase inhibitors.

Relationships between ligand binding and the shapes of the binding sites in families of homologous enzymes are investigated by comparing matrices of distances between key binding site atoms. Multiple linear regression is used to help identify key distances that influence ligand binding affinity. In order to illustrate the utility of this generic approach, we study protein kinase binding sites for ATP and the promiscuous competitive inhibitor, staurosporine. We show that the size of the gatekeeper residue and the closure between the first glycine of the GXGXXG motif and the aspartate of the DFG loop act together to promote tight binding. Our web-based tool, 'mapping analogous hetero-atoms onto residue interactions' (MAHORI), indicates that the greater the number of hydrogen bonds made by the kinase around the methylamine group of staurosporine, the tighter the binding. The conservation of surrounding atoms identified using our novel grid-based method clearly demonstrates that the most structurally conserved part of the binding site for staurosporine is the main chain of the hinge region. The critical role of interactions that are not dependent on side-chain identities is consistent with the promiscuous nature of this inhibitor.

Methods for analyzing complete gene families are becoming of increasing importance to the drug discovery process, because similarities and differences within a family are often the key to understanding functional differences that can be exploited in drug design. We undertake a large-scale structural comparison of protein kinase ATP-binding sites using a geometric hashing method. Subsequently, we propose a relevant classification of the protein kinase family based on the structural similarity of its binding sites. Our classification is not only able to reveal the great diversity of different protein kinases and therefore their different potential for inhibitor selectivity but it is also able to distinguish subtle differences within binding site conformation reflecting the protein activation state. Furthermore, using experimental inhibition profiling, we demonstrate that our classification can be used to identify protein kinase binding sites that are known experimentally to bind the same drug, demonstrating that it has potential as an inverse (protein) virtual screening tool, by identifying which other sites have the potential to bind a given drug. In this way the cross-reactivities of the anticancer drugs Tarceva and Gleevec are rationalized.

Increasingly, drug-discovery processes focus on complete gene families. Tools for analyzing similarities and differences across protein families are important for the understanding of key functional features of proteins. Herein we present a method for classifying protein families on the basis of the properties of their active sites. We have developed Cavbase, a method for describing and comparing protein binding pockets, and show its application to the functional classification of the binding pockets of the protein family of protein kinases. A diverse set of kinase cavities is mutually compared and analyzed in terms of recurring functional recognition patterns in the active sites. We are able to propose a relevant classification based on the binding motifs in the active sites. The obtained classification provides a novel perspective on functional properties across protein space. The classification of the MAP and the c-Abl kinases is analyzed in detail, showing a clear separation of the respective kinase subfamilies. Remarkable cross-relations among protein kinases are detected, in contrast to sequence-based classifications, which are not able to detect these relations. Furthermore, our classification is able to highlight features important in the optimization of protein kinase inhibitors. Using small-molecule inhibition data we could rationalize cross-reactivities between unrelated kinases which become apparent in the structural comparison of their binding sites. This procedure helps in the identification of other possible kinase targets that behave similarly in "binding pocket space" to the kinase under consideration.

Cdc37 is a molecular chaperone that has a general function in the biogenesis of protein kinases. We identified mutations within the putative “protein kinase binding domain” of Cdc37 that alleviate the conditional growth defect of a strain containing a temperature-sensitive allele, tpk2 (Ts), of the cyclic AMP-dependent protein kinase (PKA). These dominant mutations alleviate the temperature-sensitive growth defect by elevating PKA activity, as judged by their effects on PKA-regulated processes, localization and phosphorylation of the PKA effector Msn2, as well as in vitro PKA activity. Although the tpk2 (Ts) growth defect is also alleviated by Cdc37 overproduction, the CDC37 dominant mutants contain wild-type Cdc37 protein levels. In addition, Saccharomyces cerevisiae Ste11 protein kinase has an elevated physical interaction with the altered Cdc37 protein. These results implicate specific amino-terminal residues in the interaction between Cdc37 and client protein kinases and provide further genetic and biochemical support for a model in which Cdc37 functions as a molecular chaperone for protein kinases.

Mitogen‐activated protein kinases (MAPK) are broadly used regulators of cellular signaling. However, how these enzymes can be involved in such a broad spectrum of physiological functions is not understood. Systematic discovery of MAPK networks both experimentally and in silico has been hindered because MAPKs bind to other proteins with low affinity and mostly in less‐characterized disordered regions. We used a structurally consistent model on kinase‐docking motif interactions to facilitate the discovery of short functional sites in the structurally flexible and functionally under‐explored part of the human proteome and applied experimental tools specifically tailored to detect low‐affinity protein–protein interactions for their validation in vitro and in cell‐based assays. The combined computational and experimental approach enabled the identification of many novel MAPK‐docking motifs that were elusive for other large‐scale protein–protein interaction screens. The analysis produced an extensive list of independently evolved linear binding motifs from a functionally diverse set of proteins. These all target, with characteristic binding specificity, an ancient protein interaction surface on evolutionarily related but physiologically clearly distinct three MAPKs (JNK, ERK, and p38). This inventory of human protein kinase binding sites was compared with that of other organisms to examine how kinase‐mediated partnerships evolved over time. The analysis suggests that most human MAPK‐binding motifs are surprisingly new evolutionarily inventions and newly found links highlight (previously hidden) roles of MAPKs. We propose that short MAPK‐binding stretches are created in disordered protein segments through a variety of ways and they represent a major resource for ancient signaling enzymes to acquire new regulatory roles.

The c-Jun N-terminal kinase (JNK) group of mitogen-activated protein (MAP) kinases are activated by pleiotropic signals including environmental stresses, growth factors, and hormones. JNK-interacting protein 1 (JIP1) is a scaffold protein that assembles and facilitates the activation of the mixed lineage kinase-dependent JNK module and also establishes an interaction with beta-amyloid precursor protein that has been partially characterized. Here we show that, similarly to other proteins involved in various neurological diseases, JIP1 becomes hyperphosphorylated following activation of stress-activated and MAP kinases. By immobilized metal affinity chromatography and a combined microcapillary LC/MALDI-TOF/ESI-ion trap mass spectrometry approach, we identified 35 sites of mitotic phosphorylation within JIP1, among which eight were present within (Ser/Thr)-Pro sequence. This motif is modified by various kinases in aggregates of the microtubule-associated protein tau, which generates typical intraneuronal lesions occurring in Alzheimer disease. Most of the post-translational modifications found were located within the JNK, MAP kinase kinase, and RAC-alpha Ser/Thr protein kinase binding regions; no modifications occurred in protein Src homology 3 and phosphotyrosine interaction domains, which are essential for binding to kinesin, beta-amyloid precursor protein, and MAP kinase kinase kinase. Protein phosphorylation is known to affect stability and protein-protein interactions. Thus, the findings that JIP1 is extensively phosphorylated after activation of stress-activated and MAP kinases indicate that these signaling pathways might modulate JIP1 signaling by regulating its stability and association with some, but not all, interacting proteins.

Inspired by the current representation of the ligand-receptor binding process, a normal-mode-based methodology is presented to incorporate receptor flexibility in ligand docking and virtual screening. However, the systematic representation of the deformation space grows geometrically with the number of modes, and furthermore, midscale loop rearrangements like those found in protein kinase binding pockets cannot be accounted for with the first lowest-frequency modes. We thus introduced a measure of relevance of normal modes on a given region of interest and showed that only very few modes in the low-frequency range are necessary and sufficient to describe loop flexibility in cAMP-dependent protein kinase. We used this approach to generate an ensemble of representative receptor backbone conformations by perturbing the structure along a combination of relevant modes. Each ensemble conformation is complexed with known non-native binders to optimize the position of the binding-pocket side chains through a full flexible docking procedure. The multiple receptor conformations thus obtained are used in a small-scale virtual screening using receptor ensemble docking. We evaluated this algorithm on holo and apo structures of cAMP-dependent protein kinase that exhibit backbone rearrangements on two independent loop regions close to the binding pocket. Docking accuracy is improved, since the ligands considered in the virtual screening docked within 1.5 A to at least one of the structures. The discrimination between binders and nonbinders is also enhanced, as shown by the improvement of the enrichment factor. This constitutes a new step toward the systematic integration of flexible ligand-flexible receptor docking tools in structure-based drug discovery.

Hsp90 and its co-chaperone Cdc37 facilitate the folding and activation of numerous protein kinases. In this report, we examine the structure-function relationships that regulate the interaction of Cdc37 with Hsp90 and with an Hsp90-dependent kinase, the heme-regulated eIF2alpha kinase (HRI). Limited proteolysis of native and recombinant Cdc37, in conjunction with MALDI-TOF mass spectrometry analysis of peptide fragments and peptide microsequencing, indicates that Cdc37 is comprised of three discrete domains. The N-terminal domain (residues 1-126) interacts with client HRI molecules. Cdc37's middle domain (residues 128-282) interacts with Hsp90, but does not bind to HRI. The C-terminal domain of Cdc37 (residues 283-378) does not bind Hsp90 or kinase, and no functions were ascribable to this domain. Functional assays did, however, suggest that residues S127-G163 of Cdc37 serve as an interdomain switch that modulates the ability of Cdc37 to sense Hsp90's conformation and thereby mediate Hsp90's regulation of Cdc37's kinase-binding activity. Additionally, scanning alanine mutagenesis identified four amino acid residues at the N-terminus of Cdc37 that are critical for high-affinity binding of Cdc37 to client HRI molecules. One mutation, Cdc37/W7A, also implicated this region as an interpreter of Hsp90's conformation. Results illuminate the specific Cdc37 motifs underlying the allosteric interactions that regulate binding of Hsp90-Cdc37 to immature kinase molecules.

Cdc37 is a molecular chaperone required for folding of protein kinases. It functions in association with Hsp90, although little is known of its mechanism of action or where it fits into a folding pathway involving other Hsp90 cochaperones. Using a genetic approach with Saccharomyces cerevisiae, we show that CDC37 overexpression suppressed a defect in v-Src folding in yeast deleted for STI1, which recruits Hsp90 to misfolded clients. Expression of CDC37 truncation mutants that were deleted for the Hsp90-binding site stabilized v-Src and led to some folding in both sti1Δ and hsc82Δ strains. The protein kinase–binding domain of Cdc37 was sufficient for yeast cell viability and permitted efficient signaling through the yeast MAP kinase–signaling pathway. We propose a model in which Cdc37 can function independently of Hsp90, although its ability to do so is restricted by its normally low expression levels. This may be a form of regulation by which cells restrict access to Cdc37 until it has passed through a triage involving other chaperones such as Hsp70 and Hsp90.

Hepatitis B virus (HBV) controls genome encapsidation and reverse transcription from a single-stranded RNA to a double-stranded DNA through the flexible C-terminal domain (CTD) of the capsid proteins. Although the microscopic structure of the nucleocapsid plays a critical role in the life cycle of HBV, the location of CTD residues at different stages of viral replication remains poorly understood. In this work, we report the radial distributions of individual amino-acid residues of the CTD tails for both empty and RNA-containing HBV capsids by using a coarse-grained model for the key biological components and the classical density functional theory. The density functional theory calculations reveal substantial exposure of the CTD residues outside the capsid, in particular when it is devoid of any nucleic materials. The outermost layer of the capsid surface mainly consists of residues from 170Ser-175Arg of the CTD tails, i.e., the serine-arginine protein kinase binding motif. The theoretical description corroborates recent in vitro studies that show a transient CTD distribution captured by serine-arginine protein kinase binding. We have also investigated the nucleocapsid structural changes due to phosphorylation of serine residues and shown a correlation between the CTD location and the internal distribution of RNA segments.

The ultraviolet (UV) radiation resistance-associated gene (UVRAG) was initially identified for its capability to partially complement UV sensitivity in xeroderma pigmentosum (XP) cells. Subsequently, UVRAG is recognized as a tumor suppressor and autophagy-related protein. In recent years, substantial bodies of work provide new insight into UVRAG structure and multiple biological functions. UVRAG consists of a proline-rich domain, a calcium-dependent phospholipid binding C2 domain, a coiled- coil domain, a DNA-dependent protein kinase binding domain and a CEP-63 binding domain. In addition to its tumor suppression activity, UVRAG regulates autophagy and endocytic trafficking, participates in apoptosis, maintains genomic stability, gets involved in organ rotation during development. In the heart, UVRAG is required to maintain cardiac structure and function. UVRAG deficiency in the heart leads to age- related dilated cardiomyopathy. In this review, we summarize these recent findings of UVRAG that are shedding new light on its crucial roles in many biological processes and implications for the pathophysiology of various diseases including heart disease.

The magnitude and duration of signalling through mitogen- and stress-activated kinases are critical determinants of biological effect. This reflects a balance between the activities of upstream activators and a complex regulatory network of protein phosphatases. These mitogen-activated protein kinase phosphatases include both dual-specificity (threonine/tyrosine) and tyrosine-specific enzymes, and recent evidence suggests that a single mitogen-activated protein kinase isoform may be acted upon by both classes of protein phosphatase. In both cases, substrate selectivity is determined by specific protein-protein interactions mediated through noncatalytic amino-terminal mitogen-activated protein kinase binding domains. Future challenges include the determination of exactly how this network of protein phosphatases interacts selectively with mitogen-activated protein kinase signalling complexes to achieve precise regulation of these key pathways in mammalian cells.

An interaction of the hepatitis C virus (HCV) NS5A protein with the interferon (IFN)‐a‐inducible double-stranded RNA‐activated protein kinase (PKR) was demonstrated in vitro. The clinical correlation between amino acid mutations within the HCV NS5A region and response to antiviral treatment is controversial. Thirty-two patients chronically infected with HCV-1a, who were treated with IFN-a with or without ribavirin, were studied. The carboxy-terminal half of HCV NS5A was sequenced and was investigated by phylogenetic and conformational analyses. Eight patients achieved a sustained virologic response. An endof-treatment response but relapse thereafter was observed among 8 patients, whereas 16 patients were nonresponders. The median number of mutations within the PKR-binding domain but not within the previously described IFN sensitivity‐determining region was significantly higher for patients with sustained (3 mutations [range, 1‐5]) or end-of-treatment (4 mutations [range, 1‐5]) virologic response than for nonresponders (2 mutations [range, 0‐3]) ( P= ). Phylogenetic and conformational analyses of NS5A sequences allowed no differen.0087 tiation between sensitive and resistant strains. Hepatitis C virus (HCV) infection is one of the major causes of chronic hepatitis, with frequent progression to liver cirrhosis and an elevated risk for the development of hepatocellular carcinoma [1‐3]. Treatment with interferon (IFN)‐a leads to sustained virologic response, with undetectable HCV RNA in serum 24 weeks after the end of treatment in !20% of cases [4]; more recently, treatment with IFN-a in combination with ribavirin has been shown to lead to sustained virologic response in 38%‐43% of cases [5, 6]. Rates of sustained virologic response to treatment are correlated with different predictive parameters, of which HCV genotype is the strongest. In general, patients infected with HCV-2 and -3 respond better to IFN-a than do patients infected with HCV-1 subtypes [6].

PURPOSE To determine whether G-protein-mediated inhibition of secretion by met-enkephalin involves cyclic adenosine monophosphate (cAMP)-dependent events and to identify the G proteins that couple met-enkephalin to inhibition of lacrimal secretion. METHODS Secretion of protein was measured in 3-day primary cultures of rabbit lacrimal acini exposed to vehicle, the cholinergic agonist carbachol (Cch), the beta-adrenergic agonist isoproterenol (Isop), vasoactive intestinal peptide (VIP), or forskolin (FSK) with or without the enkephalin analog D-ala2-met-enkephalinamide (DALA). In separate experiments, cells were pretreated with pertussis toxin or polyclonal antibodies against the alpha subunits of Gi/Go to determine the physiologic role of G proteins in met-enkephalin inhibition of the release of lacrimal protein. Adenylyl cyclase (AC) activity was measured by a cAMP-dependent protein kinase binding assay in lacrimal membranes in response to the same agonists used in the secretion studies. RESULTS Cch resulted in a significant increase in protein release from cultured lacrimal acini. Increased secretion also occurred with Isop, VIP, and FSK. Cch- and Isop-stimulated secretion was inhibited by DALA to near-basal values. However, DALA did not inhibit VIP- or FSK-stimulated secretion. The inhibitory effect of DALA on Cch and Isop stimulation of secretion was reversed by pertussis toxin. Inhibition of Cch-stimulated secretion was blocked by antibody specific to a common peptide sequence of Gialpha1 and Gialpha2 but was not blocked by anti-Gialpha1 antibody. The inhibitory effect on Cch-stimulated secretion was also blocked by anti-Gialpha3 and anti-Goalpha. Similar experiments resulted in a reversal of DALA inhibition of beta-adrenergic stimulation of secretion by immunoneutralization of Gialpha1/2 and Goalpha but not by immunoneutralization of Gialpha1 or Gialpha3. VIP, Isop, and FSK significantly stimulated AC. However, Cch had no effect on the activity of the enzyme. In addition, DALA had no effect on AC activity under any conditions. CONCLUSIONS These results show that enkephalin inhibition of cholinergic and beta-adrenergic stimulation of secretion is mediated by Gi2, Gi3, and Go. The effector coupled by the G proteins is not AC. However, we suggest a role for met-enkephalin in G-protein-coupled modulation of ion channels important for cholinergic and beta-adrenergic stimulation of lacrimal secretion.

A-kinase anchoring proteins tether cAMP-dependent protein kinase (PKA) to specific subcellular locations. The purpose of this study was to use fluorescence resonance energy transfer to monitor binding events in living cells between the type II regulatory subunit of PKA (RII) and the RII-binding domain of the human thyroid RII anchoring protein (Ht31), a peptide containing the PKA-binding domain of an A-kinase anchoring protein. RII was linked to enhanced yellow fluorescent protein (EYFP), Ht31 was linked to enhanced cyan fluorescent protein (ECFP), and these constructs were coexpressed in Chinese hamster ovary cells. Upon excitation of the donor fluorophore, Ht31.ECFP, an increase in emission of the acceptor fluorophore, RII.EYFP, and a decrease in emission from Ht31.ECFP were observed. The emission ratio (acceptor/donor) was increased 2-fold (p0.05) in cells expressing Ht31.ECFP and RII.EYFP compared with cells expressing Ht31P.ECFP, the inactive form of Ht31, and RII.EYFP. These results provide the first in vivo demonstration of RII/Ht31 interaction in living cells and confirm previous in vitro findings of RII/Ht31 binding. Using surface plasmon resonance, we also showed that the green fluorescent protein tags did not significantly alter the binding of Ht31 to RII. Thus, fluorescence resonance energy transfer can be used to directly monitor protein-protein interactions of the PKA signaling pathway in living cells.

Cyclic AMP-dependent protein kinase is tethered to protein kinase A anchoring proteins (AKAPs) through regulatory subunits (R) by RIalpha-specific, RIIalpha-specific, or RIalpha/RIIalpha dual-specific binding. Ala- and Val-scanning mutagenesis determined that hydrophobic amino acids at three homologous positions are required for binding of RIalpha to FSC1/AKAP82 domain B and RIIalpha to AKAP Ht31. A mutation at the middle position reversed the binding specificity of both AKAPs, and mutations at this same position of the dual-specific domain A of FSC1/AKAP82 converted it into either an RIalpha or RIIalpha binding domain. This suggests that hydrophobic amino acids at three conserved positions within the primary sequence and an amphipathic helix of AKAPs are required for cyclic AMP-dependent protein kinase binding, with the size of the aliphatic side chain at the middle position determining RIalpha or RIIalpha binding specificity.

Myotonic dystrophy protein kinase (DMPK)-binding protein, MKBP, has high homology with a small heat shock protein, HSP27. Western blotting analyses showed that MKBP level in rat heart rapidly increased, with a sharp peak at one week after birth (3-fold the level at the fetus), but that it rapidly decreased (1/10 of peak value at 13 weeks). Human myocardium also showed similar age-dependency. Similar but small increase of HSP27 was observed in the neonatal rat myocardium, but not in constitutive and inducible forms of HSP70. Immunofluorescence analysis localized MKBP at the Z lines and intercalated discs in the rat myocardium. MKBP may protect actin cytoskeleton or other proteins of heart muscle against oxidative stress in the neonate.

Nopp44/46 is a phosphoprotein of the protozoan parasite Trypanosoma brucei that is localized to the nucleolus. Based on the primary sequence, Nopp44/46 appears to be a protein composed of distinct domains. This communication describes the relationship of these domains to the known functional interactions of the molecule and suggests that the amino-terminal region defines a novel homology region that functions in nucleolar targeting. We have previously shown that Nopp44/46 is capable of interacting with nucleic acids and associating with a protein kinase. Using in vitro transcription and translation, we now demonstrate that the nucleic acid binding function maps to the carboxy-terminal domain of the molecule, a region rich in arginine-glycine-glycine motifs. Our experiments reveal that a central region containing a high proportion of acidic residues is required for association with the protein kinase. Analysis of transfectants expressing epitope-tagged Nopp44/46 deletion constructs showed that the amino-terminal 96 amino acids allowed nuclear and nucleolar accumulation of the protein. This region of the molecule shows homology to several recently described nucleolar proteins. Deletion of a 27-amino-acid region within this domain abrogated nucleolar, but not nuclear, localization. These studies show that Nopp44/46 is composed of distinct modules, each of which plays a different role in molecular interactions. We suggest that this protein could facilitate interactions between sets of nucleolar molecules.

The Rat Genome Database (RGD) is the premier resource for genetic, genomic and phenotype data for the laboratory rat, Rattus norvegicus. In addition to organizing biological data from rats, the RGD team focuses on manual curation of gene–disease associations for rat, human and mouse. In this work, we have analyzed disease-associated strains, quantitative trait loci (QTL) and genes from rats. These disease objects form the basis for seven disease portals. Among disease portals, the cardiovascular disease and obesity/metabolic syndrome portals have the highest number of rat strains and QTL. These two portals share 398 rat QTL, and these shared QTL are highly concentrated on rat chromosomes 1 and 2. For disease-associated genes, we performed gene ontology (GO) enrichment analysis across portals using RatMine enrichment widgets. Fifteen GO terms, five from each GO aspect, were selected to profile enrichment patterns of each portal. Of the selected biological process (BP) terms, ‘regulation of programmed cell death’ was the top enriched term across all disease portals except in the obesity/metabolic syndrome portal where ‘lipid metabolic process’ was the most enriched term. ‘Cytosol’ and ‘nucleus’ were common cellular component (CC) annotations for disease genes, but only the cancer portal genes were highly enriched with ‘nucleus’ annotations. Similar enrichment patterns were observed in a parallel analysis using the DAVID functional annotation tool. The relationship between the preselected 15 GO terms and disease terms was examined reciprocally by retrieving rat genes annotated with these preselected terms. The individual GO term–annotated gene list showed enrichment in physiologically related diseases. For example, the ‘regulation of blood pressure’ genes were enriched with cardiovascular disease annotations, and the ‘lipid metabolic process’ genes with obesity annotations. Furthermore, we were able to enhance enrichment of neurological diseases by combining ‘G-protein coupled receptor binding’ annotated genes with ‘protein kinase binding’ annotated genes. Database URL: http://rgd.mcw.edu

Purpose In the present study, we screened a compound library containing 1,600 clinically used compounds with the aim to identify compounds, which potentially could be repositioned for colon cancer therapy. Methods Two established colon cancer cell lines were tested using the fluorometric microculture cytotoxicity assay (FMCA). For compound comparison connectivity map (CMAP) analysis, NCI 60 data mining and protein kinase binding measurements were performed. Results Sixty-eight compounds were defined as hits with activity in both of these cell lines (

A poptotic death of cardiac myocytes is a central feature of ischemic heart disease and a prime target for therapeutic intervention. Multiple stress factors are associated with ischemic stress and a battery of intrinsic pathways work to attenuate damage. These include antioxidants, antiapoptotic factors such as Bcl-2 proteins, and endogenous caspase inhibitors such as ARC (see review1). αB-crystallin is a relative newcomer to this defensive arsenal; it has strong antiapoptotic properties and, when fully induced, may constitute as much as 5% of total cardiac myocyte protein. As such, αB-crystallin may have critical structural as well as protective functions in the heart. The signaling pathways that regulate αB-crystallin activity and the molecular mechanism of protection are not understood and some aspects of these are presently controversial. In this issue of Circulation Research , Morrison et al2 describe the involvement of a critical serine residue at position 59 of the αB-crystallin protein that is targeted by the p38β-MAPKAP-K2 pathway during ischemic stress. They show that phosphorylation of this serine may be the key step for the activation and regulation of the antiapoptotic function(s) of αB-crystallin during ischemia. The crystallins are a family of dual function proteins responsible for the transparent and refractive properties of the eye lens. At least 11 vertebrate crystallins have been described and several of these have secondary chaperoning and antioxidant activities that serve to protect the lens from light-induced oxidative stress (see reviews3,4). αB-crystallin is also a small heat shock protein (smHSP) structurally related to other smHSPs including αA-crystallin, hsp27, hsp20, hsp22, myotonic dystrophy protein kinase binding protein, and HSPB3.5,6 All of these except αA-crystallin are abundantly expressed in muscle where the chaperone activity plays a key survival role in response to stress. αB-crystallin is also strongly expressed lung, kidney, brain, and heart. It …

Complicated natural products, with their structures and properties evolved over millions of years, frequently display specific biological modes of action which can often be traced back to their highly preorganized three dimensional structures that perfectly complement the shape and functional group presentation of their target protein pockets.[1] A recent study analyzed the protein binding properties of compounds from natural as well as synthetic sources and found that protein binding selectivity correlated with shape complexity (defined as the relative content of sp3-hybridized carbons) and stereochemical complexity (defined as the relative content of stereogenic carbons).[2] Octahedral metal complexes may offer an attractive alternative strategy to sophisticated globular and rigid structural templates. They are constructed from a powerful single metal stereocenter with chelating ligands limiting the degree of conformational flexibility, thus achieving “natural-product-like” structural complexities and strinkingly high target specificities as demonstrated by us in several previous studies.[3–6] An important aspect regarding the design of such metal-templated protein binders –which has not been articulated in the past– is the globular space requirement of an octahedral center. The latter significantly increases the demand for a proper design, mainly because the metal must be located at a specific position within the active site in order to be useful. For example, if the metal is located too far within the active site or too close to the protein backbone there will not be enough space available to accommodate the space-demanding octahedral coordination sphere, whereas if the metal is located too far towards the solvent, the metal center cannot easily impact on binding affinity and selectivity. A clear indicator for an advantageous metal position within the protein pocket is a strong influence of the metal coordination sphere on binding affinity and selectivity. We have identified such a priviledged position of the metal within the ATP-binding site of protein kinases by using the now well established staurosporine-inspired metallo-pyridocarbazole scaffold.[3–7] For example, the octahedral organoruthenium complex Λ-FL172 was designed as a selective inhibitor for the p21-activated kinase 1 (PAK1)[8] in which the bidentate pyridocarbazole ligand of the ruthenium complex occupies the adenine pocket.[4] By interacting with the so-called hinge region it places the ruthenium center at a defined position within the ribose binding site, where the additional CO, chloride, and bidentate iminopyridine ligands can form important contacts with other parts of the active site and thereby strongly contribute to binding affinity and selectivity (Figure 1).[4,6] In order to better understand the design of metal-based enzyme inhibitors, we wondered if this design is unique or whether other scaffolds with metals located at other positions within the active site could yield similar or even better results. To address this question, we designed new scaffolds[9] and uncovered a simple ruthenium complex (R)-1 which places the metal at a distinct position within the ATP-binding site, contains a completely different set of coordinating ligands, but at the same time shows an improved affinity for PAK1 compared to the much more complicated, previously established pyridocarbazole complex Λ-FL172. Figure 1 Complicated versus simple design for metal-templated inhibitors of the protein kinase PAK1. Ruthenium complex 1 is based on a simple pyridylphthalimide scaffold, which can be synthesized in just a few steps (Scheme 1). Accordingly, a Suzuki cross-coupling of bromophthalimide 2 with 2-trimethylstannylpyridine afforded the pyridylphthalimide 3 (49%), which was subsequently reacted with the ruthenium complex [Ru(C6H12S3)(MeCN)3](CF3SO3)2 under basic conditions, followed by the addition of NaSCN to afford the racemic complex 1 (38% over two steps). A crystal structure of the N-benzylated derivative of 1 is shown in Figure 2 and demonstrates the formation of a C-Ru bond. This cyclometallation reduces the requirement for metal-coordinating heteroatoms and is thus crucial for the short and efficient synthesis. Figure 2 Structure of the N-benzylated derivative of complex 1 (1Bn). Disordered solvent and a disordered position of S101 are not shown. ORTEP drawing with 50% probability thermal ellipsoids. Selected bond distances (A): C15-Ru1 = 2.033(4), N1-Ru1 = 2.098(3), ... Scheme 1 Synthesis of pyridylphthalimide ruthenium complex 1. The racemic complex 1 displays an IC50 value of 83 ± 20 nM (1 µM ATP) against PAK1, which is slightly more potent than the previously reported Λ-FL172 (IC50 = 130 nM, 1 µM ATP)[4] (Figure 3). Interestingly, the pyridylphthalimide ligand itself is not an inhibitor for PAK1 at all (IC50 > 100 µM), thus demonstrating the importance of the entire coordination sphere for protein kinase binding. Figure 3 IC50 curves with PAK1 of ligand 4, complex 1, and some derivative complexes 5–7. ATP concentration was 1 µM. PAK1 harbors a very atypical, open ATP-binding site which is probably responsible for the difficulties to develop high affinity inhibitors of this kinase.[4,8,10] A cocrystal structure at the resolution of 2.0 A reveals the binding of the (R)-enantiomer of 1 to the ATP binding site of PAK1 (amino acids 249–545 with mutation Lys299Arg)[11] (Figures 4 and ​and5).5). The van der Waals surface representation shown in Figure 4 demonstrates how nicely the ATP-binding site is filled with (R)-1 in a shape complementary fashion with the phthalimide moiety additionally forming two hydrogen bonds with the hinge region (Glu345 and Leu347) and one water-mediated contact to Thr406 (Figure 5). Interestingly, superimposition of this structure with the recently disclosed PAK1/Λ-FL172[4] cocrystal structure demonstrates that even though both compounds are ATP-competitive binders, they significantly differ in there binding modes (Figure 6). While both form the canonical hydrogen bonds between the maleimide moieties of the inhibitor and the hinge region of the kinase, the aromatic heterocycles are rotated towards each other by approximately 30° demonstrating the range that is possible for the directionality of hydrogen bonds. Furthermore, the metals of the two inhibitor scaffolds are 3.0 A apart from each other and point the monodentate ligands into different areas of the active site. Whereas the CO of FL172 is located right below the center of the glycine-rich loop (P-loop), the NCS ligand of 1 points towards the interface of the glycine-rich loop (connecting strands β1 and β2) and the methylene groups of Arg299 of the β-sheet strand β3 and thereby perfectly filling a small hydrophobic pocket as visualized in the van der Waals representation of Figure 4. The importance of the NCS ligand is manifested by the loss of the binding affinity of related complexes, which carry instead a SeCN (IC50 = 0.625 ± 0.06 µM, 7.5-fold weaker inhibitor), NCO (IC50 = 15.7 ± 2 µM, 193-fold weaker inhibitor), or CO (IC50 = 12.7 ± 2 µM, 153-fold weaker inhibitor) ligand (Figure 3). This intriguing example demonstrates how a different position of the metal within the active site, even if it is just shifted by 3.0 A, requires a completely different coordination sphere to fill the protein pocket in a comparable fashion. Figure 4 Cocrystal structure of PAK1 (amino acids 249–545 with mutation Lys299Arg) and (R)-1 at 2.0 A with displayed van der Waals surfaces. Coordinates of the structure have been deposited in the Protein Data Bank (PDB ID: 4DAW). Figure 5 Hydrogen bonding of (R)-1 within the ATP-binding site of PAK1. Figure 6 Relative binding position of (R)-1 and Λ-FL172 (PDB ID: 3FXZ) within the ATP-binding site of PAK1. Superimposed with the PyMOL Molecular Graphics System, Version 1.3, Schrodinger, LLC. In conclusion, the here presented metal-based enzyme inhibitor design together with the analysis of an X-ray cocrystal structure reveals the scope of fitting octahedral metal complexes within an enzyme active site. Despite the shortness of the synthesis (overall only 6 steps) and simplicity of the structure (only two stereoisomers possible), complex 1 displays an IC50 value of 83 nM (1 µM ATP) which is superior to FL172, a complex that is much more tedious to synthesize (>15 steps) and contains a much higher stereochemical complexity (20 possible stereoisomers). To date, the ruthenium phthalimide complex 1 described here belongs to the most potent ATP-competitive inhibitors known for the protein kinase PAK1,[12] demonstrating the advantages of filling large or open pockets with globular octahedral metal complexes.

Overexpression of epidermal growth factor receptor (EGFR) has been associated with cancer. Targeted inhibition of the EGFR pathway has been shown to limit proliferation of cancerous cells. Hence, we employed Traditional Chinese Medicine Database (TCM Database@Taiwan) (http://tcm.cmu.edu.tw) to identify potential EGFR inhibitor. Multiple Linear Regression (MLR), Support Vector Machine (SVM), Comparative Molecular Field Analysis (CoMFA), and Comparative Molecular Similarities Indices Analysis (CoMSIA) models were generated using a training set of EGFR ligands of known inhibitory activities. The top four TCM candidates based on DockScore were 2-O-caffeoyl tartaric acid, Emitine, Rosmaricine, and 2-O-feruloyl tartaric acid, and all had higher binding affinities than the control Iressa®. The TCM candidates had interactions with Asp855, Lys716, and Lys728, all which are residues of the protein kinase binding site. Validated MLR (r² = 0.7858) and SVM (r² = 0.8754) models predicted good bioactivity for the TCM candidates. In addition, the TCM candidates contoured well to the 3D-Quantitative Structure-Activity Relationship (3D-QSAR) map derived from the CoMFA (q² = 0.721, r² = 0.986) and CoMSIA (q² = 0.662, r² = 0.988) models. The steric field, hydrophobic field, and H-bond of the 3D-QSAR map were well matched by each TCM candidate. Molecular docking indicated that all TCM candidates formed H-bonds within the EGFR protein kinase domain. Based on the different structures, H-bonds were formed at either Asp855 or Lys716/Lys728. The compounds remained stable throughout molecular dynamics (MD) simulation. Based on the results of this study, 2-O-caffeoyl tartaric acid, Emitine, Rosmaricine, and 2-O-feruloyl tartaric acid are suggested to be potential EGFR inhibitors., National Science Council of Taiwan (NSC 99-2221-E-039-013-), Committee on Chinese Medicine and Pharmacy (CCMP100-RD-030), China Medical University (CMU98-TCM), China Medical University (CMU99-TCM), China Medical University (CMU99-S-02), China Medical University (CMU99-ASIA-25), China Medical University (CMU99-ASIA-26), China Medical University (CMU99-ASIA-27), China Medical University (CMU99-ASIA-28), Asia University, Taiwan Department of Health. Clinical Trial and Research Center of Excellence (DOH100-TD-B-111-004), Taiwan Department of Health. Cancer Research Center of Excellence (DOH100-TD-C-111-005)

We studied the alterations in binding of cyclic AMP as an indicator of particulate cyclic AMP-dependent protein kinase binding activity following transient cerebral ischemia in Mongolian gerbils and examined the effects of vinconate and pentobarbital against alterations in the binding. Animals were allowed to survive for 5 h and 7 days after 10 min of cerebral ischemia induced by bilateral occlusion of common carotid arteries. [3H]Cyclic AMP binding was significantly reduced in the hippocampus 5 h after ischemia, whereas the striatum showed no significant change in the binding. Seven days after ischemia, a severe reduction of [3H]cyclic AMP binding was noted in the dorsolateral striatum, hippocampal CA1 and CA3 sectors, and dentate gyrus. Intraperitoneal administration of vinconate (100 or 300 mg/kg) showed a significant elevation of [3H]cyclic AMP binding in the striatum, stratum pyramidale of hippocampal CA1 and CA3 sectors, and dentate gyrus 5 h after ischemia. By contrast, the intraperitoneal administration of pentobarbital (40 mg/kg) showed no significant alteration of [3H]cyclic AMP binding in most of these regions. However, vinconate and pentobarbital prevented a significant reduction of [3H]cyclic AMP binding in the dorsolateral striatum and stratum pyramidale of hippocampal CA3 sector 7 days after ischemia, although both drugs failed to prevent damage to the hippocampal CA1 sector. These results suggest that alteration in cyclic AMP binding may not be a major factor in causing ischemic neuronal damage.

Intensification of biofuel resources is urgently needed considering decreased availability of world’s fossil fuel. Palmyra palm (Borassus flabellifer) is highly potential to be developed as bioethanol source regarding the high sucrose content in its nira. It was observed that nira produced in dry season is sweeter than that in rainy season, which presumed to be influenced by a difference in expression level of sucrose-related genes during the two seasons. Study of Sucrose Synthase (SUS) gene of palmyra are therefore required prior to study of the gene expression. Palmyra SUS gene sequence is currently unavailable in GenBank, thereby pair of primers was designed from highly conserved region of SUS proteins among monocots. A 1866 bp partial cDNA fragment of SUS putative gene has been succesfully isolated from RNA of the young leaves of B. flabellifer. BLASTn and BLASTp aligments showed that either BfSUS cDNA or BfSUS polypeptide has high similarity with SUS cDNA and proteins from diverse plant species with the highest similarity shown by Tulipa gesneriana. The phylogenetic tree showed that SUS protein sequences of monocot species were distinctively grouped and splitted from those of dicot species. The BfSUS was clustered in monocot group, although not specifically grouped with particular monocot species. Nevertheless, B. flabellifer showed nearest genetic distance with Tulipa gesneriana and Oncidium cv.’Goldiana’. Characterization of BfSUS polypeptide using Geneious 4.6.2 indicated the presence of sucrose synthase (SUS) and glycosyl transferase (GT) domains, four putative UDP-glucose binding pockets within the GT domain, and a calcium-dependent Ser/Thr protein kinase binding site within the SUS domain. These domains and motifs are highly conserved in SUS proteins across plant species, confirming that the cDNA fragment obtained in this study is very likely cDNA encodes sucrose synthase in B. flabellifer.

UNLABELLED BACKGROUND. Hepatitis C virus (HCV) genotype 1 is the most prevalent genotype in Western countries, and treatment with pegylated interferon (pegIFN) plus ribavirin fails in 50%-60% of patients. Genetic variability within the NS5A dsRNA-dependent protein kinase binding domain (PKRbd) of HCV-1b has been associated with responsiveness to IFN- alpha . Little is known about NS5A sequences of HCV-1a. We investigated whether genetic variability of HCV-1a NS5A correlates with the early HCV kinetics during treatment. METHODS Twenty-four treatment-naive, HCV-1a-infected patients were treated with standard doses of pegIFN- alpha 2a plus ribavirin. HCV viremia was quantitated at days 0, 1, 2, and 3 and weeks 1, 2, 4, 8, and 12 of treatment. According to HCV kinetics, patients were classified as early rapid responders, early moderate responders, or early slow responders. The full-length HCV NS5A was sequenced at baseline and at week 1. RESULTS At baseline, variability of the NS5A C terminus (concentrated in the PKRbd) is associated with interferon efficacy but not with the second phase of the early viral decline that has been associated with a sustained virologic response. Comparisons between baseline and week-1 full-length sequences did not show significant increases in mutations. CONCLUSIONS Genetic variability of HCV-1a NS5A does not predict responsiveness to IFN- alpha . Differences in early kinetics during combination therapy are not due to selection of IFN-resistant HCV strains.

PRPK (p53-related protein kinase) has been reported as a novel protein kinase which binds to the tumor suppressor protein p53 and induces phosphorylation of p53 at Ser 15. To identify novel binding partners of PRPK, we performed a yeast two-hybrid screening and isolated an expressed sequence tag CGI-121 by which a 20-kDa protein was encoded. We demonstrated the protein–protein interaction of CGI-121 with PRPK in vivo and in vitro. The protein expression of CGI-121 was observed in many cell lines and was immunocytochemically identified in both the nucleus and cytosol. Although PRPK interacted with both CGI-121 and p53, several attempts to demonstrate an association between CGI-121 and p53 were unsuccessful. In addition, coprecipitation of p53 using recombinant PRPK was inhibited by adding recombinant CGI-121 in vitro, suggesting that CGI-121 could act as a potent inhibitor of the binding of PRPK to p53.

Background/Aims : Experimental studies have demonstrated that the wild-type PKR-NS5A strain of hepatitis C virus (HCV) may have oncogenic potential through the binding and functional repression of PKR protein kinase. To assess whether the NS5A-PKR-binding domain may be involved in HCV-related liver carcinogenesis, its sequence was analyzed in the sera of 85 patients with hepatocellular carcinoma (HCC) and in 51 patients with chronic active hepatitis (CAH). In 13 HCC cases sequence analysis was also performed in tumor and nontumor liver tissues. Methods : The nucleotide sequences of the PKR-binding domain were inferred by direct sequencing of the amplified HCV products and deduced amino acids were compared with the sequence of HCV-J. Results : A wild-type or single mutated strain which retains PKR-binding activity was found in 88% of HCC and 69% of CAH cases ( P =0.0096). All but three HCC cases showed no divergences in amino acid changes between serum and liver tissues. The wild-type strains were equally distributed between the HCC with or without cirrhosis. Conclusions : The prevalence of the wild-type NS5A-PKR strain is significantly higher in HCC than in CAH. These data suggest that inhibition of PKR activity by HCV might represent a potential mechanism of HCV-related carcinogenesis.

The small heat shock protein, human HspB2, also known as Myotonic Dystrophy Kinase Binding Protein (MKBP), specifically associates with and activates Myotonic Dystrophy Protein Kinase (DMPK), a serine/threonine protein kinase that plays an important role in maintaining muscle structure and function. The structure and function of HspB2 are not well understood. We have cloned and expressed the protein in E.coli and purified it to homogeneity. Far-UV circular dichroic spectrum of the recombinant HspB2 shows a β-sheet structure. Fluorescence spectroscopic studies show that the sole tryptophan residue at the 130(th) position is almost completely solvent-exposed. Bis-ANS binding shows that though HspB2 exhibits accessible hydrophobic surfaces, it is significantly less than that exhibited by another well characterized small HSP, αB-crystallin. Sedimentation velocity measurements show that the protein exhibits concentration-dependent oligomerization. Fluorescence resonance energy transfer study shows that HspB2 oligomers exchange subunits. Interestingly, HspB2 exhibits target protein-dependent chaperone-like activity: it exhibits significant chaperone-like activity towards dithiothreitol (DTT)-induced aggregation of insulin and heat-induced aggregation of alcohol dehydrogenase, but only partially prevents the heat-induced aggregation of citrate synthase, co-precipitating with the target protein. It also significantly prevents the ordered amyloid fibril formation of α-synuclein. Thus, our study, for the first time, provides biophysical characterization on the structural aspects of HspB2, and shows that it exhibits target protein-dependent chaperone-like activity.

Changes in the binding of [3H]cyclic AMP as an indicator of particulate cyclic AMP-dependent protein kinase (AMP-DPK) binding activity following transient forebrain ischemia were studied in the gerbil using in vitro autoradiography. [3H]Cyclic AMP binding in the strata pyramidale and lacunosum-moleculare of the hippocampal CA1, the stratum pyramidale of the CA3, and the dentate gyrus decreased transiently in the early postischemic phase but then recovered. However, [3H]cyclic AMP binding in the strata pyramidale and radiatum of the CA1, the granular layer of the dentate gyrus, and the upper layer of the cortex decreased again 7 days after ischemia. In the CA4 subfield and the lower layer of the cortex, the binding showed no significant alterations after ischemia. Administration of pentobarbital prior to the induction of ischemia prevented the decrease in [3H]cyclic AMP binding in the CA1 subfield 6 h and 7 days after ischemia, and showed protective effects against neuronal death of the CA1 pyramidal cells 7 days after ischemia. These results indicate that marked alteration of intracellular signal transduction precedes neuronal damage in the hippocampal CA1 subfield. Furthermore, postischemic reduction of [3H]cyclic AMP binding in the histologically intact cerebral cortex, CA3, and dentate gyrus may be the reflection of cellular dysfunction after energy failure.

The effects of various sugars on the simultaneous release of insulin and accumulation of cyclic AMP were studied in collagenase isolated rat pancreatic islets. D-Glucose stimulated the formation of cyclic AMP at 3 and 60 min of incubation, whether measured by a label incorporation technique, or by the protein kinase binding assay of Gilman. Only D-glucose and D-mannose were able to stimulate insulin release and cyclic [3H]AMP accumulation in the absence of other substrate. D-fructose had a stimulatory effect in the presence of 3.3 mM D-glucose only at a high concentration (33.8 mM), and enhanced the effects of 8.3 mM glucose when added at the concentration of 8.3 mM. D-Galactose was effective only together with 8.3 mM D-glucose. The order of potency of these hexoses, both regarding insulin secretion and cyclic [3H]AMP accumulation, was glucose-mannose-fructose-galactose. L-Glucose and 3-O-methylglucose had no effects at 60 min when incubated together with 8.3 mM D-glucose, whereas at 3 min, 3-O-methylglucose induced a small stimulation of the cyclic [3H]AMP response. D-mannoheptulose and D-glucosamine inhibited the insulin and cyclic [3H]AMP responses to 27.7 mM glucose. Mannoheptulose suppressed completely the glucose effect on cyclic nucleotide accumulation within 90 s. Although under all incubation conditions, the threshold stimulatory or inhibitory concentration of a given agent was identical for insulin release and cyclic [3H]AMP accumulation, these two variables showed quantitative differences in incubations of 60 min, the magnitude of the changes in insulin secretion being larger than that for the cyclic nucleotide. It is suggested that modulation of islet cyclic AMP level is an important step in the transmission of the effect of various sugars on insulin release; however, glucose and possibly other sugars may also enhance insulin release by additional mechanisms not involving the adenylate cyclase-cyclic AMP system of the beta-cell.

The effects of thymosin and lipopolysaccharide (LPS) on cyclic AMP levels in lymphocytes were evaluated using three independent assays which included adenine prelabeling, protein kinase binding, and radioimmunoassay. All three assays proved to be both sensitive and accurate in assessing relative changes in lymphocytes after incubation in vitro with various agents. The assays confirmed that basal and stimulated levels of cyclic AMP depended on the origin of the lymphocyte population. Each of the three techniques demonstrated that pyrogen-free bovine thymosin fraction 5 did not elevate thymocyte cAMP levels. In contrast, it was found that lipopolysaccharide (lps) significantly elevated cAMP levels in both spleen and thymus lymphocytes. These studies indicate that assays for measuring the activity of thymic extracts in which the intracellular levels of cyclic nucleotides are a criterion for activity are only valid if the preparations are not contaminated with endotoxins.

The phytopathogenic fungi Alternaria solani and Phymatotrichum omnivorum both contain a factor which competes with cyclic adenosine 3′,5′-monophosphate (cAMP) in the protein kinase binding assay and exhibits chromatographic behavior identical to authentic cAMP. The factor is sensitive to 3′,5′-cyclic nucleotide phosphodiesterase and has degradation kinetics identical to authentic cAMP. By these criteria the factor has been identified as cAMP. A. solani releases cAMP into the medium, and the levels of both extracellular and intracellular cAMP change during growth of A. solani in culture.

In collagenase isolated rat pancreatic islet incubates, d-glucose stimulated the accumulation of cyclic adenosine 3' : 5'-monophosphate (cAMP) as measured both by incorporation of [3H]adenine into cAMP and by assay of the total amount of nucleotide by the protein kinase binding method. l-Glucose failed to stimulate [3H]cAMP accumulation in islets. d-Glucose had no effect on [3H]cAMP accumulation in rat adipocytes. It is therefore concluded that the action of glucose is specific in the islet. Stimulation by d-glucose (5 mg per ml) was detectable after 45 to 135 s. [3H]cAMP accumulated rapidly during the first 10 min of incubation, after which a sustained but lower rate of accumulation was observed up to 120 min. 3-Isobutyl-1-methylxanthine (IBMX), 0.1 mm, markedly potentiated the stimulatory effect of a high glucose concentration, while [3H]cAMP accumulation in the presence of low glucose (0.6 mg per ml) was only slightly raised. Stimulation of cAMP accumulation induced by high glucose in the presence of IBMX was abolished within 3 min when the glucose concentration of the medium was rapidly decreased. Efflux of radioactive cyclic nucleotide was undetectable under basal conditions. In incubates stimulated by high glucose with or without IBMX, elevated contents of [3H]cAMP could be measured from 30 min on, the nucleotide continuing to accumulate in the medium up to 120 min of incubation. Over a range of glucose concentrations (0.6 to 7.0 mg per ml), insulin release after a 60-min incubation was well correlated with medium [3H]cAMP content both in the presence and absence of IBMX. Stimulation of both cAMP accumulation and insulin release were observed with small changes in medium glucose level such as increasing it from 0.9 to 1.2 mg per ml. In the incubation system used here, cAMP accumulation seems to precede the initiation of insulin release. The results presented in this report are compatible with the hypothesis that insulin release induced by glucose may be mediated by cAMP in the β-cell.

A recent study showed that cAMP analogs lowered cAMP levels in rat hepatocytes (Corbin, J.D., Beebe, S.J., and Blackmore, P.F. (1985) J. Biol. Chem. 260, 8731-8735). The present work demonstrates that cAMP analogs also lowered cAMP in a rapid, concentration-dependent manner in heart and fat cells. In order to determine if the cAMP-dependent protein kinase mediated this effect, techniques were developed to assay the protein kinase activity ratio in hepatocytes treated with cAMP analogs. The activation of protein kinase and phosphorylase in hepatocytes by 8-pCl phi S-cAMP (where 8-pCl phi S- indicates 8-parachlorothiophenyl-) was concentration-dependent and occurred in parallel to proportionate decreases in cAMP. More than 20% of the cAMP binding sites on the protein kinase were unoccupied at concentrations of 8-pCl phi S-cAMP that produced maximal cAMP lowering. Thus, the possibility that 8-pCl phi S-cAMP lowered cAMP by displacing it from protein kinase binding sites, making it available for hydrolysis, seemed unlikely. In adipocytes, the lowering of cAMP by 8-pCl phi S-cAMP occurred in parallel with increases in lipolysis and activation of low Km phosphodiesterase, suggesting that the phosphodiesterase was responsible for the cAMP lowering. Further evidence for this assertion was the finding that in hepatocytes preloaded with low concentrations of 8-pCl phi S-cAMP, glucagon lowered 8-pCl phi S-cAMP by about 50%, an amount similar to the cAMP lowering observed with 8-pCl phi S-cAMP treatment. The results were consistent with a cAMP-dependent protein kinase-catalyzed activation of a phosphodiesterase and suggested that 8-pCl phi S-cAMP-mediated hydrolysis of cAMP mimicked a physiologically significant response. The observation of this phenomenon in several tissues further suggested that it may be a general mechanism for dampening and terminating the hormonal signal through accelerated degradation of cAMP.

Arginine-rich histones H2A, H2B, H3 and H4 contain two regions of interaction with cyclic nucleotide-dependent protein kinases: a substrate phosphorylation site and a region which noncompetitively inhibits cyclic nucleotide binding to the protein kinases. We have compared the interaction of cyclic nucleotide-dependent protein kinases with these two sites in histones which are organized in nucleosome structures with the interaction of the enzymes with free histones. Whereas histones in solution are readily phosphorylated by cyclic GMP-dependent protein kinase and the catalytic subunit of cyclic AMP-dependent protein kinase, mononucleosomes are not phosphorylated by these enzymes. Histones extracted from mononucleosomes can be phosphorylated, indicating that the lack of phosphorylation of nucleosomes is not due to covalent modification of the histones but to their organization within the nucleosome structure. Whereas histones in solution are effective noncompetitive inhibitors of cyclic GMP binding to cyclic GMP-dependent protein kinase and of cyclic AMP binding to the regulatory subunits of cyclic AMP-dependent protein kinase, mononucleosomes do not affect cyclic nucleotide binding. These studies indicate that histones which are organized in nucleosome structures are neither substrates nor modifiers of cyclic nucleotide-dependent protein kinases.

The protein kinase binding assay for cAMP was modified by substitution of adsorption by QAE cellulose for the membrane filtration. This modification obviates the variation of recovery of cAMP with the volume of buffer used to wash the filter. The assay is reproducible and technically simpler than those currently employed.

Publisher Summary This chapter discusses the use of analogs to study selectivity and cooperativity of cyclic nucleotide binding sites. The chapter illustrates that cyclic AMP-dependent protein kinase holoenzyme is activated when cAMP binds to a dimeric regulatory subunit (R), and the active catalytic subunit (C) dissociates from the holoenzyme complex according to the stoichiometry addressed in the chapter. A technique is developed to study the relative selectivity of various cyclic nucleotide analogs for protein kinase binding sites. The results are applied to the obtained in site-selectivity studies to show that the binding of one class of cAMP analogs, which prefers a single intrachain site can greatly stimulate the binding of another class of site-selective analogs to the other intrachain site. This cooperativity in binding of cyclic nucleotides to both sites can be an important mechanism for amplifying the magnitude and rate of response to an in vivo hormonal elevation of intracellular cAMP levels. In addition, the methodology used to demonstrate site selectivity and cooperativity of cyclic nucleotides in binding to protein kinase can be applied to studies of ligand interactions with multiple binding sites of any receptor.