The Binding Mode of the Sonic Hedgehog Inhibitor Robotnikinin, a combined Docking and QM/MM MD Study.

NASA Astrophysics Data System (ADS)

Hitzenberger, Manuel; Schuster, Daniela; Hofer, Thomas S.

2017-10-01

Erroneous activation of the Hedgehog pathway has been linked to a great amount of cancerous diseases and therefore a large number of studies aiming at its inhibition have been carried out. One leverage point for novel therapeutic strategies targeting the proteins involved, is the prevention of complex formation between the extracellular signaling protein Sonic Hedgehog and the transmembrane protein Patched 1. In 2009 robotnikinin, a small molecule capable of binding to and inhibiting the activity of Sonic Hedgehog has been identified, however in the absence of X-ray structures of the Sonic Hedgehog-robotnikinin complex, the binding mode of this inhibitor remains unknown. In order to aid with the identification of novel Sonic Hedgehog inhibitors, the presented investigation elucidates the binding mode of robotnikinin by performing an extensive docking study, including subsequent molecular mechanical as well as quantum mechanical/molecular mechanical molecular dynamics simulations. The attained configurations enabled the identification of a number of key protein-ligand interactions, aiding complex formation and providing stabilizing contributions to the binding of the ligand. The predicted structure of the Sonic Hedgehog-robotnikinin complex is provided via a PDB file as supplementary material and can be used for further reference.

Sonic Hedgehog Guides Axons via Zipcode Binding Protein 1-Mediated Local Translation.

PubMed

Lepelletier, Léa; Langlois, Sébastien D; Kent, Christopher B; Welshhans, Kristy; Morin, Steves; Bassell, Gary J; Yam, Patricia T; Charron, Frédéric

2017-02-15

Sonic hedgehog (Shh) attracts spinal cord commissural axons toward the floorplate. How Shh elicits changes in the growth cone cytoskeleton that drive growth cone turning is unknown. We find that the turning of rat commissural axons up a Shh gradient requires protein synthesis. In particular, Shh stimulation increases β-actin protein at the growth cone even when the cell bodies have been removed. Therefore, Shh induces the local translation of β-actin at the growth cone. We hypothesized that this requires zipcode binding protein 1 (ZBP1), an mRNA-binding protein that transports β-actin mRNA and releases it for local translation upon phosphorylation. We found that Shh stimulation increases phospho-ZBP1 levels in the growth cone. Disruption of ZBP1 phosphorylation in vitro abolished the turning of commissural axons toward a Shh gradient. Disruption of ZBP1 function in vivo in mouse and chick resulted in commissural axon guidance errors. Therefore, ZBP1 is required for Shh to guide commissural axons. This identifies ZBP1 as a new mediator of noncanonical Shh signaling in axon guidance. SIGNIFICANCE STATEMENT Sonic hedgehog (Shh) guides axons via a noncanonical signaling pathway that is distinct from the canonical Hedgehog signaling pathway that specifies cell fate and morphogenesis. Axon guidance is driven by changes in the growth cone in response to gradients of guidance molecules. Little is known about the molecular mechanism of how Shh orchestrates changes in the growth cone cytoskeleton that are required for growth cone turning. Here, we show that the guidance of axons by Shh requires protein synthesis. Zipcode binding protein 1 (ZBP1) is an mRNA-binding protein that regulates the local translation of proteins, including actin, in the growth cone. We demonstrate that ZBP1 is required for Shh-mediated axon guidance, identifying a new member of the noncanonical Shh signaling pathway. Copyright © 2017 the authors 0270-6474/17/371685-11$15.00/0.

A Physical Interaction Network of Dengue Virus and Human Proteins*

PubMed Central

Khadka, Sudip; Vangeloff, Abbey D.; Zhang, Chaoying; Siddavatam, Prasad; Heaton, Nicholas S.; Wang, Ling; Sengupta, Ranjan; Sahasrabudhe, Sudhir; Randall, Glenn; Gribskov, Michael; Kuhn, Richard J.; Perera, Rushika; LaCount, Douglas J.

2011-01-01

Dengue virus (DENV), an emerging mosquito-transmitted pathogen capable of causing severe disease in humans, interacts with host cell factors to create a more favorable environment for replication. However, few interactions between DENV and human proteins have been reported to date. To identify DENV-human protein interactions, we used high-throughput yeast two-hybrid assays to screen the 10 DENV proteins against a human liver activation domain library. From 45 DNA-binding domain clones containing either full-length viral genes or partially overlapping gene fragments, we identified 139 interactions between DENV and human proteins, the vast majority of which are novel. These interactions involved 105 human proteins, including six previously implicated in DENV infection and 45 linked to the replication of other viruses. Human proteins with functions related to the complement and coagulation cascade, the centrosome, and the cytoskeleton were enriched among the DENV interaction partners. To determine if the cellular proteins were required for DENV infection, we used small interfering RNAs to inhibit their expression. Six of 12 proteins targeted (CALR, DDX3X, ERC1, GOLGA2, TRIP11, and UBE2I) caused a significant decrease in the replication of a DENV replicon. We further showed that calreticulin colocalized with viral dsRNA and with the viral NS3 and NS5 proteins in DENV-infected cells, consistent with a direct role for calreticulin in DENV replication. Human proteins that interacted with DENV had significantly higher average degree and betweenness than expected by chance, which provides additional support for the hypothesis that viruses preferentially target cellular proteins that occupy central position in the human protein interaction network. This study provides a valuable starting point for additional investigations into the roles of human proteins in DENV infection. PMID:21911577

Interactions of signaling proteins, growth factors and other proteins with heparan sulfate: mechanisms and mysteries.

PubMed

Billings, Paul C; Pacifici, Maurizio

2015-01-01

Heparan sulfate (HS) is a component of cell surface and matrix-associated proteoglycans (HSPGs) that, collectively, play crucial roles in many physiologic processes including cell differentiation, organ morphogenesis and cancer. A key function of HS is to bind and interact with signaling proteins, growth factors, plasma proteins, immune-modulators and other factors. In doing so, the HS chains and HSPGs are able to regulate protein distribution, bio-availability and action on target cells and can also serve as cell surface co-receptors, facilitating ligand-receptor interactions. These proteins contain an HS/heparin-binding domain (HBD) that mediates their association and contacts with HS. HBDs are highly diverse in sequence and predicted structure, contain clusters of basic amino acids (Lys and Arg) and possess an overall net positive charge, most often within a consensus Cardin-Weintraub (CW) motif. Interestingly, other domains and residues are now known to influence protein-HS interactions, as well as interactions with other glycosaminoglycans, such as chondroitin sulfate. In this review, we provide a description and analysis of HBDs in proteins including amphiregulin, fibroblast growth factor family members, heparanase, sclerostin and hedgehog protein family members. We discuss HBD structural and functional features and important roles carried out by other protein domains, and also provide novel conformational insights into the diversity of CW motifs present in Sonic, Indian and Desert hedgehogs. Finally, we review progress in understanding the pathogenesis of a rare pediatric skeletal disorder, Hereditary Multiple Exostoses (HME), characterized by HS deficiency and cartilage tumor formation. Advances in understanding protein-HS interactions will have broad implications for basic biology and translational medicine as well as for the development of HS-based therapeutics.

SPRINT: ultrafast protein-protein interaction prediction of the entire human interactome.

PubMed

Li, Yiwei; Ilie, Lucian

2017-11-15

Proteins perform their functions usually by interacting with other proteins. Predicting which proteins interact is a fundamental problem. Experimental methods are slow, expensive, and have a high rate of error. Many computational methods have been proposed among which sequence-based ones are very promising. However, so far no such method is able to predict effectively the entire human interactome: they require too much time or memory. We present SPRINT (Scoring PRotein INTeractions), a new sequence-based algorithm and tool for predicting protein-protein interactions. We comprehensively compare SPRINT with state-of-the-art programs on seven most reliable human PPI datasets and show that it is more accurate while running orders of magnitude faster and using very little memory. SPRINT is the only sequence-based program that can effectively predict the entire human interactome: it requires between 15 and 100 min, depending on the dataset. Our goal is to transform the very challenging problem of predicting the entire human interactome into a routine task. The source code of SPRINT is freely available from https://github.com/lucian-ilie/SPRINT/ and the datasets and predicted PPIs from www.csd.uwo.ca/faculty/ilie/SPRINT/ .

Role for protein–protein interaction databases in human genetics

PubMed Central

Pattin, Kristine A; Moore, Jason H

2010-01-01

Proteomics and the study of protein–protein interactions are becoming increasingly important in our effort to understand human diseases on a system-wide level. Thanks to the development and curation of protein-interaction databases, up-to-date information on these interaction networks is accessible and publicly available to the scientific community. As our knowledge of protein–protein interactions increases, it is important to give thought to the different ways that these resources can impact biomedical research. In this article, we highlight the importance of protein–protein interactions in human genetics and genetic epidemiology. Since protein–protein interactions demonstrate one of the strongest functional relationships between genes, combining genomic data with available proteomic data may provide us with a more in-depth understanding of common human diseases. In this review, we will discuss some of the fundamentals of protein interactions, the databases that are publicly available and how information from these databases can be used to facilitate genome-wide genetic studies. PMID:19929610

Amyloid precursor protein interaction network in human testis: sentinel proteins for male reproduction.

PubMed

Silva, Joana Vieira; Yoon, Sooyeon; Domingues, Sara; Guimarães, Sofia; Goltsev, Alexander V; da Cruz E Silva, Edgar Figueiredo; Mendes, José Fernando F; da Cruz E Silva, Odete Abreu Beirão; Fardilha, Margarida

2015-01-16

Amyloid precursor protein (APP) is widely recognized for playing a central role in Alzheimer's disease pathogenesis. Although APP is expressed in several tissues outside the human central nervous system, the functions of APP and its family members in other tissues are still poorly understood. APP is involved in several biological functions which might be potentially important for male fertility, such as cell adhesion, cell motility, signaling, and apoptosis. Furthermore, APP superfamily members are known to be associated with fertility. Knowledge on the protein networks of APP in human testis and spermatozoa will shed light on the function of APP in the male reproductive system. We performed a Yeast Two-Hybrid screen and a database search to study the interaction network of APP in human testis and sperm. To gain insights into the role of APP superfamily members in fertility, the study was extended to APP-like protein 2 (APLP2). We analyzed several topological properties of the APP interaction network and the biological and physiological properties of the proteins in the APP interaction network were also specified by gene ontologyand pathways analyses. We classified significant features related to the human male reproduction for the APP interacting proteins and identified modules of proteins with similar functional roles which may show cooperative behavior for male fertility. The present work provides the first report on the APP interactome in human testis. Our approach allowed the identification of novel interactions and recognition of key APP interacting proteins for male reproduction, particularly in sperm-oocyte interaction.

In silico prediction of protein-protein interactions in human macrophages

PubMed Central

2014-01-01

Background Protein-protein interaction (PPI) network analyses are highly valuable in deciphering and understanding the intricate organisation of cellular functions. Nevertheless, the majority of available protein-protein interaction networks are context-less, i.e. without any reference to the spatial, temporal or physiological conditions in which the interactions may occur. In this work, we are proposing a protocol to infer the most likely protein-protein interaction (PPI) network in human macrophages. Results We integrated the PPI dataset from the Agile Protein Interaction DataAnalyzer (APID) with different meta-data to infer a contextualized macrophage-specific interactome using a combination of statistical methods. The obtained interactome is enriched in experimentally verified interactions and in proteins involved in macrophage-related biological processes (i.e. immune response activation, regulation of apoptosis). As a case study, we used the contextualized interactome to highlight the cellular processes induced upon Mycobacterium tuberculosis infection. Conclusion Our work confirms that contextualizing interactomes improves the biological significance of bioinformatic analyses. More specifically, studying such inferred network rather than focusing at the gene expression level only, is informative on the processes involved in the host response. Indeed, important immune features such as apoptosis are solely highlighted when the spotlight is on the protein interaction level. PMID:24636261

The hedgehog system machinery controls transforming growth factor-β-dependent myofibroblastic differentiation in humans: involvement in idiopathic pulmonary fibrosis.

PubMed

Cigna, Natacha; Farrokhi Moshai, Elika; Brayer, Stéphanie; Marchal-Somme, Joëlle; Wémeau-Stervinou, Lidwine; Fabre, Aurélie; Mal, Hervé; Lesèche, Guy; Dehoux, Monique; Soler, Paul; Crestani, Bruno; Mailleux, Arnaud A

2012-12-01

Idiopathic pulmonary fibrosis (IPF) is a devastating disease of unknown cause. Key signaling developmental pathways are aberrantly expressed in IPF. The hedgehog pathway plays a key role during fetal lung development and may be involved in lung fibrogenesis. We determined the expression pattern of several Sonic hedgehog (SHH) pathway members in normal and IPF human lung biopsies and primary fibroblasts. The effect of hedgehog pathway inhibition was assayed by lung fibroblast proliferation and differentiation with and without transforming growth factor (TGF)-β1. We showed that the hedgehog pathway was reactivated in the IPF lung. Importantly, we deciphered the cross talk between the hedgehog and TGF-β pathway in human lung fibroblasts. TGF-β1 modulated the expression of key components of the hedgehog pathway independent of Smoothened, the obligatory signal transducer of the pathway. Smoothened was required for TGF-β1-induced myofibroblastic differentiation of control fibroblasts, but differentiation of IPF fibroblasts was partially resistant to Smoothened inhibition. Furthermore, functional hedgehog pathway machinery from the primary cilium, as well as GLI-dependent transcription in the nucleus, was required for the TGF-β1 effects on normal and IPF fibroblasts during myofibroblastic differentiation. These data identify the GLI transcription factors as potential therapeutic targets in lung fibrosis. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Identification of conserved regions and residues within Hedgehog acyltransferase critical for palmitoylation of Sonic Hedgehog.

PubMed

Buglino, John A; Resh, Marilyn D

2010-06-23

Sonic hedgehog (Shh) is a palmitoylated protein that plays key roles in mammalian development and human cancers. Palmitoylation of Shh is required for effective long and short range Shh-mediated signaling. Attachment of palmitate to Shh is catalyzed by Hedgehog acyltransferase (Hhat), a member of the membrane bound O-acyl transferase (MBOAT) family of multipass membrane proteins. The extremely hydrophobic composition of MBOAT proteins has limited their biochemical characterization. Except for mutagenesis of two conserved residues, there has been no structure-function analysis of Hhat, and the regions of the protein required for Shh palmitoylation are unknown. Here we undertake a systematic approach to identify residues within Hhat that are required for protein stability and/or enzymatic activity. We also identify a second, novel MBOAT homology region (residues 196-234) that is required for Hhat activity. In total, ten deletion mutants and eleven point mutants were generated and analyzed. Truncations at the N- and C-termini of Hhat yielded inactive proteins with reduced stability. Four Hhat mutants with deletions within predicted loop regions and five point mutants retained stability but lost palmitoylation activity. We purified two point mutants, W378A and H379A, with defective Hhat activity. Kinetic analyses revealed alterations in apparent K(m) and V(max) for Shh and/or palmitoyl CoA, changes that likely explain the catalytic defects observed for these mutants. This study has pinpointed specific regions and multiple residues that regulate Hhat stability and catalysis. Our findings should be applicable to other MBOAT proteins that mediate lipid modification of Wnt proteins and ghrelin, and should serve as a model for understanding how secreted morphogens are modified by palmitoyl acyltransferases.

Soft tissue sarcomas in the African hedgehog (Atelerix albiventris): microscopic and immunohistologic study of three cases.

PubMed

Ramos-Vara, J A

2001-09-01

Three soft tissue tumors from 2 female hedgehogs were examined microscopically and immunohistochemically. Two tumors involved haired skin and the third one was vaginal. Microscopically, the cutaneous tumors had features of malignant peripheral nerve sheath tumor (MPNST), whereas the vaginal tumor was classified only as a spindle cell sarcoma. Immunohistochemically, all 3 tumors were strongly positive for vimentin and strongly to moderately positive for CD10 and neuron-specific enolase but did not stain with antibody to S100 protein, an antigen typically present in human MPNST The cutaneous tumor from hedgehog no. 1 was examined ultrastructurally and the neoplastic cells resembled fibroblasts. Hedgehog no. 1 was euthanized at the time of the biopsy. The outcome of the other hedgehog was unknown.

Desert hedgehog is a mammal-specific gene expressed during testicular and ovarian development in a marsupial.

PubMed

O'Hara, William A; Azar, Walid J; Behringer, Richard R; Renfree, Marilyn B; Pask, Andrew J

2011-12-01

Desert hedgehog (DHH) belongs to the hedgehog gene family that act as secreted intercellular signal transducers. DHH is an essential morphogen for normal testicular development and function in both mice and humans but is not present in the avian lineage. Like other hedgehog proteins, DHH signals through the patched (PTCH) receptors 1 and 2. Here we examine the expression and protein distribution of DHH, PTCH1 and PTCH2 in the developing testes of a marsupial mammal (the tammar wallaby) to determine whether DHH signalling is a conserved factor in gonadal development in all therian mammals. DHH, PTCH1 and PTCH2 were present in the marsupial genome and highly conserved with their eutherian orthologues. Phylogenetic analyses indicate that DHH has recently evolved and is a mammal-specific hedgehog orthologue. The marsupial PTCH2 receptor had an additional exon (exon 21a) not annotated in eutherian PTCH2 proteins. Interestingly we found evidence of this exon in humans and show that its translation would result in a truncated protein with functions similar to PTCH1. We also show that DHH expression was not restricted to the testes during gonadal development (as in mice), but was also expressed in the developing ovary. Expression of DHH, PTCH1 and PTCH2 in the adult tammar testis and ovary was consistent with findings in the adult mouse. These data suggest that there is a highly conserved role for DHH signalling in the differentiation and function of the mammalian testis and that DHH may be necessary for marsupial ovarian development. The receptors PTCH1 and PTCH2 are highly conserved mediators of hedgehog signalling in both the developing and adult marsupial gonads. Together these findings indicate DHH is an essential therian mammal-specific morphogen in gonadal development and gametogenesis.

Loss of function mutation in the palmitoyl-transferase HHAT leads to syndromic 46,XY disorder of sex development by impeding Hedgehog protein palmitoylation and signaling.

PubMed

Callier, Patrick; Calvel, Pierre; Matevossian, Armine; Makrythanasis, Periklis; Bernard, Pascal; Kurosaka, Hiroshi; Vannier, Anne; Thauvin-Robinet, Christel; Borel, Christelle; Mazaud-Guittot, Séverine; Rolland, Antoine; Desdoits-Lethimonier, Christèle; Guipponi, Michel; Zimmermann, Céline; Stévant, Isabelle; Kuhne, Françoise; Conne, Béatrice; Santoni, Federico; Lambert, Sandy; Huet, Frederic; Mugneret, Francine; Jaruzelska, Jadwiga; Faivre, Laurence; Wilhelm, Dagmar; Jégou, Bernard; Trainor, Paul A; Resh, Marilyn D; Antonarakis, Stylianos E; Nef, Serge

2014-05-01

The Hedgehog (Hh) family of secreted proteins act as morphogens to control embryonic patterning and development in a variety of organ systems. Post-translational covalent attachment of cholesterol and palmitate to Hh proteins are critical for multimerization and long range signaling potency. However, the biological impact of lipid modifications on Hh ligand distribution and signal reception in humans remains unclear. In the present study, we report a unique case of autosomal recessive syndromic 46,XY Disorder of Sex Development (DSD) with testicular dysgenesis and chondrodysplasia resulting from a homozygous G287V missense mutation in the hedgehog acyl-transferase (HHAT) gene. This mutation occurred in the conserved membrane bound O-acyltransferase (MBOAT) domain and experimentally disrupted the ability of HHAT to palmitoylate Hh proteins such as DHH and SHH. Consistent with the patient phenotype, HHAT was found to be expressed in the somatic cells of both XX and XY gonads at the time of sex determination, and Hhat loss of function in mice recapitulates most of the testicular, skeletal, neuronal and growth defects observed in humans. In the developing testis, HHAT is not required for Sertoli cell commitment but plays a role in proper testis cord formation and the differentiation of fetal Leydig cells. Altogether, these results shed new light on the mechanisms of action of Hh proteins. Furthermore, they provide the first clinical evidence of the essential role played by lipid modification of Hh proteins in human testicular organogenesis and embryonic development.

Loss of Function Mutation in the Palmitoyl-Transferase HHAT Leads to Syndromic 46,XY Disorder of Sex Development by Impeding Hedgehog Protein Palmitoylation and Signaling

PubMed Central

Makrythanasis, Periklis; Bernard, Pascal; Kurosaka, Hiroshi; Vannier, Anne; Thauvin-Robinet, Christel; Borel, Christelle; Mazaud-Guittot, Séverine; Rolland, Antoine; Desdoits-Lethimonier, Christèle; Guipponi, Michel; Zimmermann, Céline; Stévant, Isabelle; Kuhne, Françoise; Conne, Béatrice; Santoni, Federico; Lambert, Sandy; Huet, Frederic; Mugneret, Francine; Jaruzelska, Jadwiga; Faivre, Laurence; Wilhelm, Dagmar; Jégou, Bernard; Trainor, Paul A.; Resh, Marilyn D.; Antonarakis, Stylianos E.; Nef, Serge

2014-01-01

The Hedgehog (Hh) family of secreted proteins act as morphogens to control embryonic patterning and development in a variety of organ systems. Post-translational covalent attachment of cholesterol and palmitate to Hh proteins are critical for multimerization and long range signaling potency. However, the biological impact of lipid modifications on Hh ligand distribution and signal reception in humans remains unclear. In the present study, we report a unique case of autosomal recessive syndromic 46,XY Disorder of Sex Development (DSD) with testicular dysgenesis and chondrodysplasia resulting from a homozygous G287V missense mutation in the hedgehog acyl-transferase (HHAT) gene. This mutation occurred in the conserved membrane bound O-acyltransferase (MBOAT) domain and experimentally disrupted the ability of HHAT to palmitoylate Hh proteins such as DHH and SHH. Consistent with the patient phenotype, HHAT was found to be expressed in the somatic cells of both XX and XY gonads at the time of sex determination, and Hhat loss of function in mice recapitulates most of the testicular, skeletal, neuronal and growth defects observed in humans. In the developing testis, HHAT is not required for Sertoli cell commitment but plays a role in proper testis cord formation and the differentiation of fetal Leydig cells. Altogether, these results shed new light on the mechanisms of action of Hh proteins. Furthermore, they provide the first clinical evidence of the essential role played by lipid modification of Hh proteins in human testicular organogenesis and embryonic development. PMID:24784881

Signatures of pleiotropy, economy and convergent evolution in a domain-resolved map of human-virus protein-protein interaction networks.

PubMed

Garamszegi, Sara; Franzosa, Eric A; Xia, Yu

2013-01-01

A central challenge in host-pathogen systems biology is the elucidation of general, systems-level principles that distinguish host-pathogen interactions from within-host interactions. Current analyses of host-pathogen and within-host protein-protein interaction networks are largely limited by their resolution, treating proteins as nodes and interactions as edges. Here, we construct a domain-resolved map of human-virus and within-human protein-protein interaction networks by annotating protein interactions with high-coverage, high-accuracy, domain-centric interaction mechanisms: (1) domain-domain interactions, in which a domain in one protein binds to a domain in a second protein, and (2) domain-motif interactions, in which a domain in one protein binds to a short, linear peptide motif in a second protein. Analysis of these domain-resolved networks reveals, for the first time, significant mechanistic differences between virus-human and within-human interactions at the resolution of single domains. While human proteins tend to compete with each other for domain binding sites by means of sequence similarity, viral proteins tend to compete with human proteins for domain binding sites in the absence of sequence similarity. Independent of their previously established preference for targeting human protein hubs, viral proteins also preferentially target human proteins containing linear motif-binding domains. Compared to human proteins, viral proteins participate in more domain-motif interactions, target more unique linear motif-binding domains per residue, and contain more unique linear motifs per residue. Together, these results suggest that viruses surmount genome size constraints by convergently evolving multiple short linear motifs in order to effectively mimic, hijack, and manipulate complex host processes for their survival. Our domain-resolved analyses reveal unique signatures of pleiotropy, economy, and convergent evolution in viral-host interactions that are

Gas1 extends the range of Hedgehog action by facilitating its signaling

PubMed Central

Martinelli, David C.; Fan, Chen-Ming

2007-01-01

Cellular signaling initiated by Hedgehog binding to Patched1 has profound importance in mammalian embryogenesis, genetic disease, and cancer. Hedgehog acts as a morphogen to specify distinctive cell fates using different concentration thresholds, but our knowledge of how the concentration gradient is interpreted into the activity gradient is incomplete. The membrane protein Growth Arrest-Specific Gene 1 (GAS1) was thought to be a negative regulator of the Hedgehog concentration gradient. Here, we report unexpected genetic evidence that Gas1 positively regulates Hedgehog signaling in multiple developmental contexts, an effect particularly noticeable at regions where Hedgehog acts at low concentration. Using a combination of in vitro cell culture and in ovo electroporation assays, we demonstrate that GAS1 acts cooperatively with Patched1 for Hedgehog binding and enhances signaling activity in a cell-autonomous manner. Our data support a model in which GAS1 helps transform the Hedgehog protein gradient into the observed activity gradient. We propose that Gas1 is an evolutionarily novel, vertebrate-specific Hedgehog pathway regulator. PMID:17504940

Aberrant Epithelial-Mesenchymal Hedgehog Signaling Characterizes Barrett's Metaplasia

PubMed Central

Wang, David H.; Clemons, Nicholas J.; Miyashita, Tomoharu; Dupuy, Adam J.; Zhang, Wei; Szczepny, Anette; Corcoran-Schwartz, Ian M.; Wilburn, Daniel L.; Montgomery, Elizabeth A.; Wang, Jean S.; Jenkins, Nancy A.; Copeland, Neal A.; Harmon, John W.; Phillips, Wayne A.; Watkins, D. Neil

2010-01-01

Background & Aims The molecular mechanism underlying epithelial metaplasia in Barrett's esophagus remains unknown. Recognizing that Hedgehog signaling is required for early esophageal development, we sought to determine if the Hedgehog pathway is reactivated in Barrett's esophagus, and if genes downstream of the pathway could promote columnar differentiation of esophageal epithelium. Methods Immunohistochemistry, immunofluorescence, and quantitative real-time PCR were used to analyze clinical specimens, human esophageal cell lines, and mouse esophagi. Human esophageal squamous epithelial (HET-1A) and adenocarcinoma (OE33) cells were subjected to acid treatment and used in transfection experiments. Swiss Webster mice were used in a surgical model of bile reflux injury. An in vivo transplant culture system was created using esophageal epithelium from Sonic hedgehog transgenic mice. Results Marked upregulation of Hedgehog ligand expression, which can be induced by acid or bile exposure, occurs frequently in Barrett's epithelium and is associated with stromal expression of the Hedgehog target genes PTCH1 and BMP4. BMP4 signaling induces expression of SOX9, an intestinal crypt transcription factor, which is highly expressed in Barrett's epithelium. We further show that expression of DMBT1, the human homologue of the columnar cell factor Hensin, occurs in Barrett's epithelium and is induced by SOX9. Finally, transgenic expression of Sonic hedgehog in mouse esophageal epithelium induces expression of stromal Bmp4, epithelial Sox9 and columnar cytokeratins. Conclusions Epithelial Hedgehog ligand expression may contribute to the initiation of Barrett's esophagus through induction of stromal BMP4, which triggers reprogramming of esophageal epithelium in favor of a columnar phenotype. PMID:20138038

Insight into bacterial virulence mechanisms against host immune response via the Yersinia pestis-human protein-protein interaction network.

PubMed

Yang, Huiying; Ke, Yuehua; Wang, Jian; Tan, Yafang; Myeni, Sebenzile K; Li, Dong; Shi, Qinghai; Yan, Yanfeng; Chen, Hui; Guo, Zhaobiao; Yuan, Yanzhi; Yang, Xiaoming; Yang, Ruifu; Du, Zongmin

2011-11-01

A Yersinia pestis-human protein interaction network is reported here to improve our understanding of its pathogenesis. Up to 204 interactions between 66 Y. pestis bait proteins and 109 human proteins were identified by yeast two-hybrid assay and then combined with 23 previously published interactions to construct a protein-protein interaction network. Topological analysis of the interaction network revealed that human proteins targeted by Y. pestis were significantly enriched in the proteins that are central in the human protein-protein interaction network. Analysis of this network showed that signaling pathways important for host immune responses were preferentially targeted by Y. pestis, including the pathways involved in focal adhesion, regulation of cytoskeleton, leukocyte transendoepithelial migration, and Toll-like receptor (TLR) and mitogen-activated protein kinase (MAPK) signaling. Cellular pathways targeted by Y. pestis are highly relevant to its pathogenesis. Interactions with host proteins involved in focal adhesion and cytoskeketon regulation pathways could account for resistance of Y. pestis to phagocytosis. Interference with TLR and MAPK signaling pathways by Y. pestis reflects common characteristics of pathogen-host interaction that bacterial pathogens have evolved to evade host innate immune response by interacting with proteins in those signaling pathways. Interestingly, a large portion of human proteins interacting with Y. pestis (16/109) also interacted with viral proteins (Epstein-Barr virus [EBV] and hepatitis C virus [HCV]), suggesting that viral and bacterial pathogens attack common cellular functions to facilitate infections. In addition, we identified vasodilator-stimulated phosphoprotein (VASP) as a novel interaction partner of YpkA and showed that YpkA could inhibit in vitro actin assembly mediated by VASP.

Hedgehog hives.

PubMed

Fairley, J A; Suchniak, J; Paller, A S

1999-05-01

Hedgehogs are increasingly popular pets in the United States and Europe. A number of infections may be acquired from these animals, and hedgehogs are possible hosts of parasites. However, to our knowledge there arc no previous reports of urticarial reactions to hedgehogs. We describe 3 patients who developed an acute, transient, urticarial reaction after contact with the extended spines of pet hedgehogs. One patient also developed a more prolonged reaction at the site of contact. Interestingly, all 3 patients had documented allergies to cats and/or dogs. The results of prick testing in 1 patient to an extract of hedgehog dander produced an immediate wheal-and-flare reaction. A variety of dermatologic disorders may be seen in handlers of hedgehogs. Due to the increasing popularity of these animals as pets, it is likely that these reactions will be noted more frequently by dermatologists. The presence of allergies to other pets may be predictive of hedgehog hives and further investigation of the cross reaction of various animal antigens may clarify this relationship.

The Hedgehog Signal Transduction Network

PubMed Central

Robbins, David J.; Fei, Dennis Liang; Riobo, Natalia A.

2013-01-01

Hedgehog (Hh) proteins regulate the development of a wide range of metazoan embryonic and adult structures, and disruption of Hh signaling pathways results in various human diseases. Here, we provide a comprehensive review of the signaling pathways regulated by Hh, consolidating data from a diverse array of organisms in a variety of scientific disciplines. Similar to the elucidation of many other signaling pathways, our knowledge of Hh signaling developed in a sequential manner centered on its earliest discoveries. Thus, our knowledge of Hh signaling has for the most part focused on elucidating the mechanism by which Hh regulates the Gli family of transcription factors, the so-called “canonical” Hh signaling pathway. However, in the past few years, numerous studies have shown that Hh proteins can also signal through Gli-independent mechanisms collectively referred to as “noncanonical” signaling pathways. Noncanonical Hh signaling is itself subdivided into two distinct signaling modules: (i) those not requiring Smoothened (Smo) and (ii) those downstream of Smo that do not require Gli transcription factors. Thus, Hh signaling is now proposed to occur through a variety of distinct context-dependent signaling modules that have the ability to crosstalk with one another to form an interacting, dynamic Hh signaling network. PMID:23074268

Motile cilia of human airway epithelia contain hedgehog signaling components that mediate noncanonical hedgehog signaling.

PubMed

Mao, Suifang; Shah, Alok S; Moninger, Thomas O; Ostedgaard, Lynda S; Lu, Lin; Tang, Xiao Xiao; Thornell, Ian M; Reznikov, Leah R; Ernst, Sarah E; Karp, Philip H; Tan, Ping; Keshavjee, Shaf; Abou Alaiwa, Mahmoud H; Welsh, Michael J

2018-02-06

Differentiated airway epithelia produce sonic hedgehog (SHH), which is found in the thin layer of liquid covering the airway surface. Although previous studies showed that vertebrate HH signaling requires primary cilia, as airway epithelia mature, the cells lose primary cilia and produce hundreds of motile cilia. Thus, whether airway epithelia have apical receptors for SHH has remained unknown. We discovered that motile cilia on airway epithelial cells have HH signaling proteins, including patched and smoothened. These cilia also have proteins affecting cAMP-dependent signaling, including Gα i and adenylyl cyclase 5/6. Apical SHH decreases intracellular levels of cAMP, which reduces ciliary beat frequency and pH in airway surface liquid. These results suggest that apical SHH may mediate noncanonical HH signaling through motile cilia to dampen respiratory defenses at the contact point between the environment and the lung, perhaps counterbalancing processes that stimulate airway defenses. Copyright © 2018 the Author(s). Published by PNAS.

Cryptosporidium erinacei n. sp. (Apicomplexa: Cryptosporidiidae) in hedgehogs.

PubMed

Kváč, Martin; Hofmannová, Lada; Hlásková, Lenka; Květoňová, Dana; Vítovec, Jiří; McEvoy, John; Sak, Bohumil

2014-03-17

The morphological, biological, and molecular characteristics of Cryptosporidium hedgehog genotype are described, and the species name Cryptosporidium erinacei n. sp. is proposed to reflect its specificity for hedgehogs under natural and experimental conditions. Oocysts of C. erinacei are morphologically indistinguishable from Cryptosporidium parvum, measuring 4.5-5.8 μm (mean=4.9 μm) × 4.0-4.8 μm (mean=4.4 μm) with a length to width ratio of 1.13 (1.02-1.35) (n=100). Oocysts of C. erinacei obtained from a naturally infected European hedgehog (Erinaceus europaeus) were infectious for naïve 8-week-old four-toed hedgehogs (Atelerix albiventris); the prepatent period was 4-5 days post infection (DPI) and the patent period was longer than 20 days. C. erinacei was not infectious for 8-week-old SCID and BALB/c mice (Mus musculus), Mongolian gerbils (Meriones unguiculatus), or golden hamsters (Mesocricetus auratus). Phylogenetic analyses based on small subunit rRNA, 60 kDa glycoprotein, actin, Cryptosporidium oocyst wall protein, thrombospondin-related adhesive protein of Cryptosporidium-1, and heat shock protein 70 gene sequences revealed that C. erinacei is genetically distinct from previously described Cryptosporidium species. Copyright © 2014 Elsevier B.V. All rights reserved.

Multiple protein-protein interactions converging on the Prp38 protein during activation of the human spliceosome.

PubMed

Schütze, Tonio; Ulrich, Alexander K C; Apelt, Luise; Will, Cindy L; Bartlick, Natascha; Seeger, Martin; Weber, Gert; Lührmann, Reinhard; Stelzl, Ulrich; Wahl, Markus C

2016-02-01

Spliceosomal Prp38 proteins contain a conserved amino-terminal domain, but only higher eukaryotic orthologs also harbor a carboxy-terminal RS domain, a hallmark of splicing regulatory SR proteins. We show by crystal structure analysis that the amino-terminal domain of human Prp38 is organized around three pairs of antiparallel α-helices and lacks similarities to RNA-binding domains found in canonical SR proteins. Instead, yeast two-hybrid analyses suggest that the amino-terminal domain is a versatile protein-protein interaction hub that possibly binds 12 other spliceosomal proteins, most of which are recruited at the same stage as Prp38. By quantitative, alanine surface-scanning two-hybrid screens and biochemical analyses we delineated four distinct interfaces on the Prp38 amino-terminal domain. In vitro interaction assays using recombinant proteins showed that Prp38 can bind at least two proteins simultaneously via two different interfaces. Addition of excess Prp38 amino-terminal domain to in vitro splicing assays, but not of an interaction-deficient mutant, stalled splicing at a precatalytic stage. Our results show that human Prp38 is an unusual SR protein, whose amino-terminal domain is a multi-interface protein-protein interaction platform that might organize the relative positioning of other proteins during splicing. © 2016 Schütze et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Hedgehog Signaling in Pancreatic Fibrosis and Cancer

PubMed Central

Bai, Yongyu; Bai, Yongheng; Dong, Jiaojiao; Li, Qiang; Jin, Yuepeng; Chen, Bicheng; Zhou, Mengtao

2016-01-01

Abstract The hedgehog signaling pathway was first discovered in the 1980s. It is a stem cell-related pathway that plays a crucial role in embryonic development, tissue regeneration, and organogenesis. Aberrant activation of hedgehog signaling leads to pathological consequences, including a variety of human tumors such as pancreatic cancer. Multiple lines of evidence indicate that blockade of this pathway with several small-molecule inhibitors can inhibit the development of pancreatic neoplasm. In addition, activated hedgehog signaling has been reported to be involved in fibrogenesis in many tissues, including the pancreas. Therefore, new therapeutic targets based on hedgehog signaling have attracted a great deal of attention to alleviate pancreatic diseases. In this review, we briefly discuss the recent advances in hedgehog signaling in pancreatic fibrogenesis and carcinogenesis and highlight new insights on their potential relationship with respect to the development of novel targeted therapies. PMID:26962810

Hedgehog Pathway Inhibition Radiosensitizes Non-Small Cell Lung Cancers

PubMed Central

Zeng, Jing; Aziz, Khaled; Chettiar, Sivarajan T.; Aftab, Blake T.; Armour, Michael; Gajula, Rajendra; Gandhi, Nishant; Salih, Tarek; Herman, Joseph M.; Wong, John; Rudin, Charles M.; Tran, Phuoc T.; Hales, Russell K.

2012-01-01

Purpose Despite improvements in chemoradiation, local control remains a major clinical problem in locally advanced non-small cell lung cancer. The Hedgehog pathway has been implicated in tumor recurrence by promoting survival of tumorigenic precursors and through effects on tumor-associated stroma. Whether Hedgehog inhibition can affect radiation efficacy in vivo has not been reported. Methods and Materials We evaluated the effects of a targeted Hedgehog inhibitor (HhAntag) and radiation on clonogenic survival of human non-small cell lung cancer lines in vitro. Using an A549 cell line xenograft model, we examined tumor growth, proliferation, apoptosis, and gene expression changes after concomitant HhAntag and radiation. In a transgenic mouse model of KrasG12D-induced and Twist1-induced lung adenocarcinoma, we assessed tumor response to radiation and HhAntag by serial micro-computed tomography (CT) scanning. Results In 4 human lung cancer lines in vitro, HhAntag showed little or no effect on radio-sensitivity. By contrast, in both the human tumor xenograft and murine inducible transgenic models, HhAntag enhanced radiation efficacy and delayed tumor growth. By use of the human xenograft model to differentiate tumor and stromal effects, mouse stromal cells, but not human tumor cells, showed significant and consistent downregulation of Hedgehog pathway gene expression. This was associated with increased tumor cell apoptosis. Conclusions Targeted Hedgehog pathway inhibition can increase in vivo radiation efficacy in lung cancer preclinical models. This effect is associated with pathway suppression in tumor-associated stroma. These data support clinical testing of Hedgehog inhibitors as a component of multimodality therapy for locally advanced non-small cell lung cancer. PMID:23182391

Hedgehog pathway inhibition radiosensitizes non-small cell lung cancers.

PubMed

Zeng, Jing; Aziz, Khaled; Chettiar, Sivarajan T; Aftab, Blake T; Armour, Michael; Gajula, Rajendra; Gandhi, Nishant; Salih, Tarek; Herman, Joseph M; Wong, John; Rudin, Charles M; Tran, Phuoc T; Hales, Russell K

2013-05-01

Despite improvements in chemoradiation, local control remains a major clinical problem in locally advanced non-small cell lung cancer. The Hedgehog pathway has been implicated in tumor recurrence by promoting survival of tumorigenic precursors and through effects on tumor-associated stroma. Whether Hedgehog inhibition can affect radiation efficacy in vivo has not been reported. We evaluated the effects of a targeted Hedgehog inhibitor (HhAntag) and radiation on clonogenic survival of human non-small cell lung cancer lines in vitro. Using an A549 cell line xenograft model, we examined tumor growth, proliferation, apoptosis, and gene expression changes after concomitant HhAntag and radiation. In a transgenic mouse model of Kras(G12D)-induced and Twist1-induced lung adenocarcinoma, we assessed tumor response to radiation and HhAntag by serial micro-computed tomography (CT) scanning. In 4 human lung cancer lines in vitro, HhAntag showed little or no effect on radiosensitivity. By contrast, in both the human tumor xenograft and murine inducible transgenic models, HhAntag enhanced radiation efficacy and delayed tumor growth. By use of the human xenograft model to differentiate tumor and stromal effects, mouse stromal cells, but not human tumor cells, showed significant and consistent downregulation of Hedgehog pathway gene expression. This was associated with increased tumor cell apoptosis. Targeted Hedgehog pathway inhibition can increase in vivo radiation efficacy in lung cancer preclinical models. This effect is associated with pathway suppression in tumor-associated stroma. These data support clinical testing of Hedgehog inhibitors as a component of multimodality therapy for locally advanced non-small cell lung cancer. Copyright © 2013 Elsevier Inc. All rights reserved.

Hedgehog Pathway Inhibition Radiosensitizes Non-Small Cell Lung Cancers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zeng, Jing; Aziz, Khaled; Chettiar, Sivarajan T.

2013-05-01

Purpose: Despite improvements in chemoradiation, local control remains a major clinical problem in locally advanced non-small cell lung cancer. The Hedgehog pathway has been implicated in tumor recurrence by promoting survival of tumorigenic precursors and through effects on tumor-associated stroma. Whether Hedgehog inhibition can affect radiation efficacy in vivo has not been reported. Methods and Materials: We evaluated the effects of a targeted Hedgehog inhibitor (HhAntag) and radiation on clonogenic survival of human non-small cell lung cancer lines in vitro. Using an A549 cell line xenograft model, we examined tumor growth, proliferation, apoptosis, and gene expression changes after concomitant HhAntagmore » and radiation. In a transgenic mouse model of Kras{sup G12D}-induced and Twist1-induced lung adenocarcinoma, we assessed tumor response to radiation and HhAntag by serial micro-computed tomography (CT) scanning. Results: In 4 human lung cancer lines in vitro, HhAntag showed little or no effect on radiosensitivity. By contrast, in both the human tumor xenograft and murine inducible transgenic models, HhAntag enhanced radiation efficacy and delayed tumor growth. By use of the human xenograft model to differentiate tumor and stromal effects, mouse stromal cells, but not human tumor cells, showed significant and consistent downregulation of Hedgehog pathway gene expression. This was associated with increased tumor cell apoptosis. Conclusions: Targeted Hedgehog pathway inhibition can increase in vivo radiation efficacy in lung cancer preclinical models. This effect is associated with pathway suppression in tumor-associated stroma. These data support clinical testing of Hedgehog inhibitors as a component of multimodality therapy for locally advanced non-small cell lung cancer.« less

New partner proteins containing novel internal recognition motif for human Glutaminase Interacting Protein (hGIP)

PubMed Central

Zencir, Sevil; Banerjee, Monimoy; Dobson, Melanie J.; Ayaydin, Ferhan; Fodor, Elfrieda Ayaydin; Topcu, Zeki; Mohanty, Smita

2013-01-01

Regulation of gene expression in cells is mediated by protein-protein, DNA-protein and receptor-ligand interactions. PDZ (PSD-95/Discs-large/ZO-1) domains are protein–protein interaction modules. PDZ-containing proteins function in the organization of multi-protein complexes controlling spatial and temporal fidelity of intracellular signaling pathways. In general, PDZ proteins possess multiple domains facilitating distinct interactions. The human Glutaminase Interacting Protein (hGIP) is an unusual PDZ protein comprising entirely of a single PDZ domain and plays pivotal roles in many cellular processes through its interaction with the C-terminus of partner proteins. Here, we report the identification by yeast two-hybrid screening of two new hGIP-interacting partners, DTX1 and STAU1. Both proteins lack the typical C-terminal PDZ recognition motif but contain a novel internal hGIP recognition motif recently identified in a phage display library screen. Fluorescence resonance energy transfer and confocal microscopy analysis confirmed the in vivo association of hGIP with DTX1 and STAU1 in mammalian cells validating the previous discovery of S/T-X-V/L-D as a consensus internal motif for hGIP recognition. Similar to hGIP, DTX1 and STAU1 have been implicated in neuronal function. Identification of these new interacting partners furthers our understanding of GIP-regulated signaling cascades and these interactions may represent potential new drug targets in humans. PMID:23395680

Active Sonic Hedgehog Signaling between Androgen Independent Human Prostate Cancer Cells and Normal/Benign but Not Cancer-Associated Prostate Stromal Cells

PubMed Central

Shigemura, Katsumi; Huang, Wen-Chin; Li, Xiangyan; Zhau, Haiyen E.; Zhu, Guodong; Gotoh, Akinobu; Fujisawa, Masato; Xie, Jingwu; Marshall, Fray F.; Chung, Leland W. K.

2012-01-01

BACKGROUND Sonic hedgehog (Shh) signaling plays a pivotal role in stromal-epithelial interaction during normal development but its role in tumor-stromal interaction during carcinogenic progression is less well defined. Since hormone refractory prostate cancer with bone metastasis is difficult to treat, it is crucial to investigate how androgen independent (AI) human prostate cancer cells communicate with their associated stroma. METHODS Shh and its target transcription factor, Gli1 mRNA, were assessed by RT-PCR and/or quantitative RT-PCR in co-cultured cell recombinants comprised of AI C4-2 either with NPF (prostate fibroblasts from normal/benign prostate gland) or CPF cancer-associated stromal fibroblasts) under Shh/cyclopamine (a hedgehog signaling inhibitor) treatment. Human bone marrow stromal (HS27A) cells were used as controls. In vivo investigation was performed by checking serum PSA and immunohistochemical staining for the apoptosis-associated M30 gene in mice bearing chimeric C4-2/NPF tumors. RESULTS CONCLUSIONS Based on co-culture and chimeric tumor models, active Shh-mediated signaling was demonstrated between AI prostate cancer and NPF in a paracrine- and tumor progression-dependent manner. Our study suggests that drugs like cyclopamine that interfere with Shh signaling could be beneficial in preventing AI progression in prostate cancer cells. PMID:21520153

Prediction of the Ebola Virus Infection Related Human Genes Using Protein-Protein Interaction Network.

PubMed

Cao, HuanHuan; Zhang, YuHang; Zhao, Jia; Zhu, Liucun; Wang, Yi; Li, JiaRui; Feng, Yuan-Ming; Zhang, Ning

2017-01-01

Ebola hemorrhagic fever (EHF) is caused by Ebola virus (EBOV). It is reported that human could be infected by EBOV with a high fatality rate. However, association factors between EBOV and host still tend to be ambiguous. According to the "guilt by association" (GBA) principle, proteins interacting with each other are very likely to function similarly or the same. Based on this assumption, we tried to obtain EBOV infection-related human genes in a protein-protein interaction network using Dijkstra algorithm. We hope it could contribute to the discovery of novel effective treatments. Finally, 15 genes were selected as potential EBOV infection-related human genes. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

hPDI: a database of experimental human protein-DNA interactions.

PubMed

Xie, Zhi; Hu, Shaohui; Blackshaw, Seth; Zhu, Heng; Qian, Jiang

2010-01-15

The human protein DNA Interactome (hPDI) database holds experimental protein-DNA interaction data for humans identified by protein microarray assays. The unique characteristics of hPDI are that it contains consensus DNA-binding sequences not only for nearly 500 human transcription factors but also for >500 unconventional DNA-binding proteins, which are completely uncharacterized previously. Users can browse, search and download a subset or the entire data via a web interface. This database is freely accessible for any academic purposes. http://bioinfo.wilmer.jhu.edu/PDI/.

Efficient prediction of human protein-protein interactions at a global scale.

PubMed

Schoenrock, Andrew; Samanfar, Bahram; Pitre, Sylvain; Hooshyar, Mohsen; Jin, Ke; Phillips, Charles A; Wang, Hui; Phanse, Sadhna; Omidi, Katayoun; Gui, Yuan; Alamgir, Md; Wong, Alex; Barrenäs, Fredrik; Babu, Mohan; Benson, Mikael; Langston, Michael A; Green, James R; Dehne, Frank; Golshani, Ashkan

2014-12-10

Our knowledge of global protein-protein interaction (PPI) networks in complex organisms such as humans is hindered by technical limitations of current methods. On the basis of short co-occurring polypeptide regions, we developed a tool called MP-PIPE capable of predicting a global human PPI network within 3 months. With a recall of 23% at a precision of 82.1%, we predicted 172,132 putative PPIs. We demonstrate the usefulness of these predictions through a range of experiments. The speed and accuracy associated with MP-PIPE can make this a potential tool to study individual human PPI networks (from genomic sequences alone) for personalized medicine.

Hedgehog Zoonoses

PubMed Central

Riley, Patricia Y.

2005-01-01

Exotic pets, including hedgehogs, have become popular in recent years among pet owners, especially in North America. Such animals can carry and introduce zoonotic agents, a fact well illustrated by the recent outbreak of monkeypox in pet prairie dogs. We reviewed known and potential zoonotic diseases that could be carried and transmitted by pet hedgehogs or when rescuing and caring for wild-caught hedgehogs. PMID:15705314

Engineering Archeal Surrogate Systems for the Development of Protein-Protein Interaction Inhibitors against Human RAD51.

PubMed

Moschetti, Tommaso; Sharpe, Timothy; Fischer, Gerhard; Marsh, May E; Ng, Hong Kin; Morgan, Matthew; Scott, Duncan E; Blundell, Tom L; R Venkitaraman, Ashok; Skidmore, John; Abell, Chris; Hyvönen, Marko

2016-11-20

Protein-protein interactions (PPIs) are increasingly important targets for drug discovery. Efficient fragment-based drug discovery approaches to tackle PPIs are often stymied by difficulties in the production of stable, unliganded target proteins. Here, we report an approach that exploits protein engineering to "humanise" thermophilic archeal surrogate proteins as targets for small-molecule inhibitor discovery and to exemplify this approach in the development of inhibitors against the PPI between the recombinase RAD51 and tumour suppressor BRCA2. As human RAD51 has proved impossible to produce in a form that is compatible with the requirements of fragment-based drug discovery, we have developed a surrogate protein system using RadA from Pyrococcus furiosus. Using a monomerised RadA as our starting point, we have adopted two parallel and mutually instructive approaches to mimic the human enzyme: firstly by mutating RadA to increase sequence identity with RAD51 in the BRC repeat binding sites, and secondly by generating a chimeric archaeal human protein. Both approaches generate proteins that interact with a fourth BRC repeat with affinity and stoichiometry comparable to human RAD51. Stepwise humanisation has also allowed us to elucidate the determinants of RAD51 binding to BRC repeats and the contributions of key interacting residues to this interaction. These surrogate proteins have enabled the development of biochemical and biophysical assays in our ongoing fragment-based small-molecule inhibitor programme and they have allowed us to determine hundreds of liganded structures in support of our structure-guided design process, demonstrating the feasibility and advantages of using archeal surrogates to overcome difficulties in handling human proteins. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Activation of the Hedgehog Signaling Pathway in the Developing Lens Stimulates Ectopic FoxE3 Expression and Disruption in Fiber Cell Differentiation

PubMed Central

Kerr, Christine L.; Huang, Jian; Williams, Trevor; West-Mays, Judith A.

2012-01-01

Purpose. The signaling pathways and transcriptional effectors responsible for directing mammalian lens development provide key regulatory molecules that can inform our understanding of human eye defects. The hedgehog genes encode extracellular signaling proteins responsible for patterning and tissue formation during embryogenesis. Signal transduction of this pathway is mediated through activation of the transmembrane proteins smoothened and patched, stimulating downstream signaling resulting in the activation or repression of hedgehog target genes. Hedgehog signaling is implicated in eye development, and defects in hedgehog signaling components have been shown to result in defects of the retina, iris, and lens. Methods. We assessed the consequences of constitutive hedgehog signaling in the developing mouse lens using Cre-LoxP technology to express the conditional M2 smoothened allele in the embryonic head and lens ectoderm. Results. Although initial lens development appeared normal, morphological defects were apparent by E12.5 and became more significant at later stages of embryogenesis. Altered lens morphology correlated with ectopic expression of FoxE3, which encodes a critical gene required for human and mouse lens development. Later, inappropriate expression of the epithelial marker Pax6, and as well as fiber cell markers c-maf and Prox1 also occurred, indicating a failure of appropriate lens fiber cell differentiation accompanied by altered lens cell proliferation and cell death. Conclusions. Our findings demonstrate that the ectopic activation of downstream effectors of the hedgehog signaling pathway in the mouse lens disrupts normal fiber cell differentiation by a mechanism consistent with a sustained epithelial cellular developmental program driven by FoxE3. PMID:22491411

Genetic interactions between the hedgehog co-receptors Gas1 and Boc regulate cell proliferation during murine palatogenesis

PubMed Central

Xavier, Guilherme M.; Seppala, Maisa; Papageorgiou, Spyridon N.; Fan, Chen-Ming; Cobourne, Martyn T.

2016-01-01

Abnormal regulation of Sonic hedgehog (Shh) signaling has been described in a variety of human cancers and developmental anomalies, which highlights the essential role of this signaling molecule in cell cycle regulation and embryonic development. Gas1 and Boc are membrane co-receptors for Shh, which demonstrate overlapping domains of expression in the early face. This study aims to investigate potential interactions between these co-receptors during formation of the secondary palate. Mice with targeted mutation in Gas1 and Boc were used to generate Gas1; Boc compound mutants. The expression of key Hedgehog signaling family members was examined in detail during palatogenesis via radioactive in situ hybridization. Morphometric analysis involved computational quantification of BrdU-labeling and cell packing; whilst TUNEL staining was used to assay cell death. Ablation of Boc in a Gas1 mutant background leads to reduced Shh activity in the palatal shelves and an increase in the penetrance and severity of cleft palate, associated with failed elevation, increased proliferation and reduced cell death. Our findings suggest a dual requirement for Boc and Gas1 during early development of the palate, mediating cell cycle regulation during growth and subsequent fusion of the palatal shelves. PMID:27811357

Computational prediction of virus-human protein-protein interactions using embedding kernelized heterogeneous data.

PubMed

Nourani, Esmaeil; Khunjush, Farshad; Durmuş, Saliha

2016-05-24

Pathogenic microorganisms exploit host cellular mechanisms and evade host defense mechanisms through molecular pathogen-host interactions (PHIs). Therefore, comprehensive analysis of these PHI networks should be an initial step for developing effective therapeutics against infectious diseases. Computational prediction of PHI data is gaining increasing demand because of scarcity of experimental data. Prediction of protein-protein interactions (PPIs) within PHI systems can be formulated as a classification problem, which requires the knowledge of non-interacting protein pairs. This is a restricting requirement since we lack datasets that report non-interacting protein pairs. In this study, we formulated the "computational prediction of PHI data" problem using kernel embedding of heterogeneous data. This eliminates the abovementioned requirement and enables us to predict new interactions without randomly labeling protein pairs as non-interacting. Domain-domain associations are used to filter the predicted results leading to 175 novel PHIs between 170 human proteins and 105 viral proteins. To compare our results with the state-of-the-art studies that use a binary classification formulation, we modified our settings to consider the same formulation. Detailed evaluations are conducted and our results provide more than 10 percent improvements for accuracy and AUC (area under the receiving operating curve) results in comparison with state-of-the-art methods.

Identification of proteins that may directly interact with human RPA.

PubMed

Nakaya, Ryou; Takaya, Junichiro; Onuki, Takeshi; Moritani, Mariko; Nozaki, Naohito; Ishimi, Yukio

2010-11-01

RPA, which consisted of three subunits (RPA1, 2 and 3), plays essential roles in DNA transactions. At the DNA replication forks, RPA binds to single-stranded DNA region to stabilize the structure and to assemble other replication proteins. Interactions between RPA and several replication proteins have been reported but the analysis is not comprehensive. We systematically performed the qualitative analysis to identify RPA interaction partners to understand the protein-protein interaction at the replication forks. We expressed in insect cells the three subunits of human RPA, together with one replication protein, which is present at the forks under normal conditions and/or under the replication stress conditions, to examine the interaction. Among 30 proteins examined in total, it was found that at least 14 proteins interacted with RPA. RPA interacted with MCM3-7, MCM-BP and CDC45 proteins among the proteins that play roles in the initiation and the elongation of the DNA replication. RPA bound with TIPIN, CLASPIN and RAD17, which are involved in the DNA replication checkpoint functions. RPA also bound with cyclin-dependent kinases and an amino-terminal fragment of Rb protein that negatively regulates DNA replication. These results suggest that RPA interacts with the specific proteins among those that play roles in the regulation of the replication fork progression.

SARS coronavirus protein 7a interacts with human Ap4A-hydrolase.

PubMed

Vasilenko, Natalia; Moshynskyy, Igor; Zakhartchouk, Alexander

2010-02-09

The SARS coronavirus (SARS-CoV) open reading frame 7a (ORF 7a) encodes a 122 amino acid accessory protein. It has no significant sequence homology with any other known proteins. The 7a protein is present in the virus particle and has been shown to interact with several host proteins; thereby implicating it as being involved in several pathogenic processes including apoptosis, inhibition of cellular protein synthesis, and activation of p38 mitogen activated protein kinase. In this study we present data demonstrating that the SARS-CoV 7a protein interacts with human Ap4A-hydrolase (asymmetrical diadenosine tetraphosphate hydrolase, EC 3.6.1.17). Ap4A-hydrolase is responsible for metabolizing the "allarmone" nucleotide Ap4A and therefore likely involved in regulation of cell proliferation, DNA replication, RNA processing, apoptosis and DNA repair. The interaction between 7a and Ap4A-hydrolase was identified using yeast two-hybrid screening. The interaction was confirmed by co-immunoprecipitation from cultured human cells transiently expressing V5-His tagged 7a and HA tagged Ap4A-hydrolase. Human tissue culture cells transiently expressing 7a and Ap4A-hydrolase tagged with EGFP and Ds-Red2 respectively show these proteins co-localize in the cytoplasm.

Human germline hedgehog pathway mutations predispose to fatty liver.

PubMed

Guillen-Sacoto, Maria J; Martinez, Ariel F; Abe, Yu; Kruszka, Paul; Weiss, Karin; Everson, Joshua L; Bataller, Ramon; Kleiner, David E; Ward, Jerrold M; Sulik, Kathleen K; Lipinski, Robert J; Solomon, Benjamin D; Muenke, Maximilian

2017-10-01

Non-alcoholic fatty liver disease (NAFLD) is the most common form of liver disease. Activation of hedgehog (Hh) signaling has been implicated in the progression of NAFLD and proposed as a therapeutic target; however, the effects of Hh signaling inhibition have not been studied in humans with germline mutations that affect this pathway. Patients with holoprosencephaly (HPE), a disorder associated with germline mutations disrupting Sonic hedgehog (SHH) signaling, were clinically evaluated for NAFLD. A combined mouse model of Hh signaling attenuation (Gli2 heterozygous null: Gli2 +/- ) and diet-induced NAFLD was used to examine aspects of NAFLD and hepatic gene expression profiles, including molecular markers of hepatic fibrosis and inflammation. Patients with HPE had a higher prevalence of liver steatosis compared to the general population, independent of obesity. Exposure of Gli2 +/- mice to fatty liver-inducing diets resulted in increased liver steatosis compared to wild-type mice. Similar to humans, this effect was independent of obesity in the mutant mice and was associated with decreased expression of pro-fibrotic and pro-inflammatory genes, and increased expression of PPARγ, a potent anti-fibrogenic and anti-inflammatory regulator. Interestingly, tumor suppressors p53 and p16INK4 were found to be downregulated in the Gli2 +/- mice exposed to a high-fat diet. Our results indicate that germline mutations disrupting Hh signaling promotes liver steatosis, independent of obesity, with reduced fibrosis. While Hh signaling inhibition has been associated with a better NAFLD prognosis, further studies are required to evaluate the long-term effects of mutations affecting this pathway. Lay summary: Non-alcoholic fatty liver disease (NAFLD) is characterized by excess fat deposition in the liver predominantly due to high calorie intake and a sedentary lifestyle. NAFLD progression is usually accompanied by activation of the Sonic hedgehog (SHH) pathway leading to fibrous

Sonic Hedgehog Protein Is Decreased and Penile Morphology Is Altered in Prostatectomy and Diabetic Patients

PubMed Central

Angeloni, Nicholas L.; Bond, Christopher W.; McVary, Kevin T.; Podlasek, Carol A.

2013-01-01

Erectile dysfunction (ED) is a debilitating medical condition and current treatments are ineffective in patients with cavernous nerve (CN) injury, due to penile remodeling and apoptosis. A critical regulator of penile smooth muscle and apoptosis is the secreted protein sonic hedgehog (SHH). SHH protein is decreased in rat prostatectomy and diabetic ED models, SHH inhibition in the penis induces apoptosis and ED, and SHH treatment at the time of CN injury suppresses smooth muscle apoptosis and promotes regeneration of erectile function. Thus SHH treatment has significant translational potential as an ED therapy if similar mechanisms underlie ED development in patients. In this study we quantify SHH protein and morphological changes in corpora cavernosal tissue of control, prostatectomy and diabetic patients and hypothesize that decreased SHH protein is an underlying cause of ED development in prostatectomy and diabetic patients. Our results show significantly decreased SHH protein in prostatectomy and diabetic penis. Morphological remodelling of the penis, including significantly increased apoptotic index and decreased smooth muscle/collagen ratio, accompanies declining SHH. SHH signaling is active in human penis and is altered in a parallel manner to previous observations in the rat. These results suggest that SHH has significant potential to be developed as an ED therapy in prostatectomy and diabetic patients. The increased apoptotic index long after initial injury is suggestive of ongoing remodeling that may be clinically manipulatable. PMID:23967143

Astrocytoma in an African hedgehog (Atelerix albiventris) suspected wobbly hedgehog syndrome.

PubMed

Nakata, Makoto; Miwa, Yasutsugu; Itou, Takuya; Uchida, Kazuyuki; Nakayama, Hiroyuki; Sakai, Takeo

2011-10-01

A 28-month-old African hedgehog was referred to our hospital with progressive tetraparesis. On the first presentation, the hedgehog was suspected as having wobbly hedgehog syndrome (WHS) and the animal was treated with medication and rehabilitation. The animal died 22 days after onset. Pathological examination revealed that the animal was involved in astrocytoma between the medulla oblongata and the spinal cord (C1). This report indicates that a primary central nervous system tumor should be considered as one of the differential diagnoses for hedgehogs presenting with progressive paresis, together with WHS.

RAID: a comprehensive resource for human RNA-associated (RNA–RNA/RNA–protein) interaction

PubMed Central

Zhang, Xiaomeng; Wu, Deng; Chen, Liqun; Li, Xiang; Yang, Jinxurong; Fan, Dandan; Dong, Tingting; Liu, Mingyue; Tan, Puwen; Xu, Jintian; Yi, Ying; Wang, Yuting; Zou, Hua; Hu, Yongfei; Fan, Kaili; Kang, Juanjuan; Huang, Yan; Miao, Zhengqiang; Bi, Miaoman; Jin, Nana; Li, Kongning; Li, Xia; Xu, Jianzhen; Wang, Dong

2014-01-01

Transcriptomic analyses have revealed an unexpected complexity in the eukaryote transcriptome, which includes not only protein-coding transcripts but also an expanding catalog of noncoding RNAs (ncRNAs). Diverse coding and noncoding RNAs (ncRNAs) perform functions through interaction with each other in various cellular processes. In this project, we have developed RAID (http://www.rna-society.org/raid), an RNA-associated (RNA–RNA/RNA–protein) interaction database. RAID intends to provide the scientific community with all-in-one resources for efficient browsing and extraction of the RNA-associated interactions in human. This version of RAID contains more than 6100 RNA-associated interactions obtained by manually reviewing more than 2100 published papers, including 4493 RNA–RNA interactions and 1619 RNA–protein interactions. Each entry contains detailed information on an RNA-associated interaction, including RAID ID, RNA/protein symbol, RNA/protein categories, validated method, expressing tissue, literature references (Pubmed IDs), and detailed functional description. Users can query, browse, analyze, and manipulate RNA-associated (RNA–RNA/RNA–protein) interaction. RAID provides a comprehensive resource of human RNA-associated (RNA–RNA/RNA–protein) interaction network. Furthermore, this resource will help in uncovering the generic organizing principles of cellular function network. PMID:24803509

Multiple protein–protein interactions converging on the Prp38 protein during activation of the human spliceosome

PubMed Central

Schütze, Tonio; Ulrich, Alexander K.C.; Apelt, Luise; Will, Cindy L.; Bartlick, Natascha; Seeger, Martin; Weber, Gert; Lührmann, Reinhard; Stelzl, Ulrich; Wahl, Markus C.

2016-01-01

Spliceosomal Prp38 proteins contain a conserved amino-terminal domain, but only higher eukaryotic orthologs also harbor a carboxy-terminal RS domain, a hallmark of splicing regulatory SR proteins. We show by crystal structure analysis that the amino-terminal domain of human Prp38 is organized around three pairs of antiparallel α-helices and lacks similarities to RNA-binding domains found in canonical SR proteins. Instead, yeast two-hybrid analyses suggest that the amino-terminal domain is a versatile protein–protein interaction hub that possibly binds 12 other spliceosomal proteins, most of which are recruited at the same stage as Prp38. By quantitative, alanine surface-scanning two-hybrid screens and biochemical analyses we delineated four distinct interfaces on the Prp38 amino-terminal domain. In vitro interaction assays using recombinant proteins showed that Prp38 can bind at least two proteins simultaneously via two different interfaces. Addition of excess Prp38 amino-terminal domain to in vitro splicing assays, but not of an interaction-deficient mutant, stalled splicing at a precatalytic stage. Our results show that human Prp38 is an unusual SR protein, whose amino-terminal domain is a multi-interface protein–protein interaction platform that might organize the relative positioning of other proteins during splicing. PMID:26673105

Multivalency of Sonic hedgehog conjugated to linear polymer chains modulates protein potency.

PubMed

Wall, Samuel T; Saha, Krishanu; Ashton, Randolph S; Kam, Kimberly R; Schaffer, David V; Healy, Kevin E

2008-04-01

A potently active multivalent form of the protein Sonic hedgehog (Shh) was produced by bioconjugation of a modified recombinant form of Shh to the linear polymers poly(acrylic acid) (pAAc) and hyaluronic acid (HyA) via a two-step reaction exploiting carboimiide and maleimide chemistry. Efficiency of the conjugation was approximately 75% even at stoichiometric ratios of 30 Shh molecules per linear HyA chain (i.e., 30:1 Shh/HyA). Bioactivity of the conjugates was tested via a cellular assay across a range of stoichiometric ratios of Shh molecules to HyA linear chains, which was varied from 0.6:1 Shh/HyA to 22:1 Shh/HyA. Results indicate that low conjugation ratios decrease Shh bioactivity and high ratios increase this activity beyond the potency of monomeric Shh, with approximately equal activity between monomeric soluble Shh and conjugated Shh at 7:1 Shh/HyA. In addition, high-ratio constructs increased angiogenesis determined by the in vivo chick chorioallantoic membrane (CAM) assay. These results are captured by a kinetic model of multiple interactions between the Shh/HyA conjugates and cell surface receptors resulting in higher cell signaling at lower bulk Shh concentrations.

RAID: a comprehensive resource for human RNA-associated (RNA-RNA/RNA-protein) interaction.

PubMed

Zhang, Xiaomeng; Wu, Deng; Chen, Liqun; Li, Xiang; Yang, Jinxurong; Fan, Dandan; Dong, Tingting; Liu, Mingyue; Tan, Puwen; Xu, Jintian; Yi, Ying; Wang, Yuting; Zou, Hua; Hu, Yongfei; Fan, Kaili; Kang, Juanjuan; Huang, Yan; Miao, Zhengqiang; Bi, Miaoman; Jin, Nana; Li, Kongning; Li, Xia; Xu, Jianzhen; Wang, Dong

2014-07-01

Transcriptomic analyses have revealed an unexpected complexity in the eukaryote transcriptome, which includes not only protein-coding transcripts but also an expanding catalog of noncoding RNAs (ncRNAs). Diverse coding and noncoding RNAs (ncRNAs) perform functions through interaction with each other in various cellular processes. In this project, we have developed RAID (http://www.rna-society.org/raid), an RNA-associated (RNA-RNA/RNA-protein) interaction database. RAID intends to provide the scientific community with all-in-one resources for efficient browsing and extraction of the RNA-associated interactions in human. This version of RAID contains more than 6100 RNA-associated interactions obtained by manually reviewing more than 2100 published papers, including 4493 RNA-RNA interactions and 1619 RNA-protein interactions. Each entry contains detailed information on an RNA-associated interaction, including RAID ID, RNA/protein symbol, RNA/protein categories, validated method, expressing tissue, literature references (Pubmed IDs), and detailed functional description. Users can query, browse, analyze, and manipulate RNA-associated (RNA-RNA/RNA-protein) interaction. RAID provides a comprehensive resource of human RNA-associated (RNA-RNA/RNA-protein) interaction network. Furthermore, this resource will help in uncovering the generic organizing principles of cellular function network. © 2014 Zhang et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Mapping Protein Interactions between Dengue Virus and Its Human and Insect Hosts

PubMed Central

Doolittle, Janet M.; Gomez, Shawn M.

2011-01-01

Background Dengue fever is an increasingly significant arthropod-borne viral disease, with at least 50 million cases per year worldwide. As with other viral pathogens, dengue virus is dependent on its host to perform the bulk of functions necessary for viral survival and replication. To be successful, dengue must manipulate host cell biological processes towards its own ends, while avoiding elimination by the immune system. Protein-protein interactions between the virus and its host are one avenue through which dengue can connect and exploit these host cellular pathways and processes. Methodology/Principal Findings We implemented a computational approach to predict interactions between Dengue virus (DENV) and both of its hosts, Homo sapiens and the insect vector Aedes aegypti. Our approach is based on structural similarity between DENV and host proteins and incorporates knowledge from the literature to further support a subset of the predictions. We predict over 4,000 interactions between DENV and humans, as well as 176 interactions between DENV and A. aegypti. Additional filtering based on shared Gene Ontology cellular component annotation reduced the number of predictions to approximately 2,000 for humans and 18 for A. aegypti. Of 19 experimentally validated interactions between DENV and humans extracted from the literature, this method was able to predict nearly half (9). Additional predictions suggest specific interactions between virus and host proteins relevant to interferon signaling, transcriptional regulation, stress, and the unfolded protein response. Conclusions/Significance Dengue virus manipulates cellular processes to its advantage through specific interactions with the host's protein interaction network. The interaction networks presented here provide a set of hypothesis for further experimental investigation into the DENV life cycle as well as potential therapeutic targets. PMID:21358811

Hedgehog signaling in the murine melanoma microenvironment.

PubMed

Geng, Ling; Cuneo, Kyle C; Cooper, Michael K; Wang, Hong; Sekhar, Konjeti; Fu, Allie; Hallahan, Dennis E

2007-01-01

The Hedgehog intercellular signaling pathway regulates cell proliferation and differentiation. This pathway has been implicated to play a role in the pathogenesis of cancer and in embryonic blood vessel development. In the current study, Hedgehog signaling in tumor related vasculature and microenvironment was examined using human umbilical vein endothelial cells and B16F0 (murine melanoma) tumors models. Use of exogenous Sonic hedgehog (Shh) peptide significantly increased BrdU incorporation in endothelial cells in vitro by a factor of 2 (P < 0.001). The Hedgehog pathway antagonist cyclopamine effectively reduced Shh-induced proliferation to control levels. To study Hedgehog signaling in vivo a hind limb tumor model with the B16F0 cell line was used. Treatment with 25 mg/kg cyclopamine significantly attenuated BrdU incorporation in tumor cells threefold (P < 0.001), in tumor related endothelial cells threefold (P = 0.004), and delayed tumor growth by 4 days. Immunohistochemistry revealed that the Hedgehog receptor Patched was localized to the tumor stroma and that B16F0 cells expressed Shh peptide. Furthermore, mouse embryonic fibroblasts required the presence of B16F0 cells to express Patched in a co-culture assay system. These studies indicate that Shh peptide produced by melanoma cells induces Patched expression in fibroblasts. To study tumor related angiogenesis a vascular window model was used to monitor tumor vascularity. Treatment with cyclopamine significantly attenuated vascular formation by a factor of 2.5 (P < 0.001) and altered vascular morphology. Furthermore, cyclopamine reduced tumor blood vessel permeability to FITC labeled dextran while having no effect on normal blood vessels. These studies suggest that Hedgehog signaling regulates melanoma related vascular formation and function.

Defining the Protein–Protein Interaction Network of the Human Hippo Pathway*

PubMed Central

Wang, Wenqi; Li, Xu; Huang, Jun; Feng, Lin; Dolinta, Keithlee G.; Chen, Junjie

2014-01-01

The Hippo pathway, which is conserved from Drosophila to mammals, has been recognized as a tumor suppressor signaling pathway governing cell proliferation and apoptosis, two key events involved in organ size control and tumorigenesis. Although several upstream regulators, the conserved kinase cascade and key downstream effectors including nuclear transcriptional factors have been defined, the global organization of this signaling pathway is not been fully understood. Thus, we conducted a proteomic analysis of human Hippo pathway, which revealed the involvement of an extensive protein–protein interaction network in this pathway. The mass spectrometry data were deposited to ProteomeXchange with identifier PXD000415. Our data suggest that 550 interactions within 343 unique protein components constitute the central protein–protein interaction landscape of human Hippo pathway. Our study provides a glimpse into the global organization of Hippo pathway, reveals previously unknown interactions within this pathway, and uncovers new potential components involved in the regulation of this pathway. Understanding these interactions will help us further dissect the Hippo signaling-pathway and extend our knowledge of organ size control. PMID:24126142

A Novel Biclustering Approach to Association Rule Mining for Predicting HIV-1–Human Protein Interactions

PubMed Central

Mukhopadhyay, Anirban; Maulik, Ujjwal; Bandyopadhyay, Sanghamitra

2012-01-01

Identification of potential viral-host protein interactions is a vital and useful approach towards development of new drugs targeting those interactions. In recent days, computational tools are being utilized for predicting viral-host interactions. Recently a database containing records of experimentally validated interactions between a set of HIV-1 proteins and a set of human proteins has been published. The problem of predicting new interactions based on this database is usually posed as a classification problem. However, posing the problem as a classification one suffers from the lack of biologically validated negative interactions. Therefore it will be beneficial to use the existing database for predicting new viral-host interactions without the need of negative samples. Motivated by this, in this article, the HIV-1–human protein interaction database has been analyzed using association rule mining. The main objective is to identify a set of association rules both among the HIV-1 proteins and among the human proteins, and use these rules for predicting new interactions. In this regard, a novel association rule mining technique based on biclustering has been proposed for discovering frequent closed itemsets followed by the association rules from the adjacency matrix of the HIV-1–human interaction network. Novel HIV-1–human interactions have been predicted based on the discovered association rules and tested for biological significance. For validation of the predicted new interactions, gene ontology-based and pathway-based studies have been performed. These studies show that the human proteins which are predicted to interact with a particular viral protein share many common biological activities. Moreover, literature survey has been used for validation purpose to identify some predicted interactions that are already validated experimentally but not present in the database. Comparison with other prediction methods is also discussed. PMID:22539940

Hedgehog regulates Norrie disease protein to drive neural progenitor self-renewal.

PubMed

McNeill, Brian; Mazerolle, Chantal; Bassett, Erin A; Mears, Alan J; Ringuette, Randy; Lagali, Pamela; Picketts, David J; Paes, Kim; Rice, Dennis; Wallace, Valerie A

2013-03-01

Norrie disease (ND) is a congenital disorder characterized by retinal hypovascularization and cognitive delay. ND has been linked to mutations in 'Norrie Disease Protein' (Ndp), which encodes the secreted protein Norrin. Norrin functions as a secreted angiogenic factor, although its role in neural development has not been assessed. Here, we show that Ndp expression is initiated in retinal progenitors in response to Hedgehog (Hh) signaling, which induces Gli2 binding to the Ndp promoter. Using a combination of genetic epistasis and acute RNAi-knockdown approaches, we show that Ndp is required downstream of Hh activation to induce retinal progenitor proliferation in the retina. Strikingly, Ndp regulates the rate of cell-cycle re-entry and not cell-cycle kinetics, thereby uncoupling the self-renewal and cell-cycle progression functions of Hh. Taken together, we have uncovered a cell autonomous function for Ndp in retinal progenitor proliferation that is independent of its function in the retinal vasculature, which could explain the neural defects associated with ND.

PM2.5 promotes human bronchial smooth muscle cell migration via the sonic hedgehog signaling pathway.

PubMed

Ye, Xiuqin; Hong, Wei; Hao, Binwei; Peng, Gongyong; Huang, Lingmei; Zhao, Zhuxiang; Zhou, Yumin; Zheng, Mengning; Li, Chenglong; Liang, Chunxiao; Yi, Erkang; Pu, Jinding; Li, Bing; Ran, Pixin

2018-03-02

The contribution of airway remodeling in chronic obstructive pulmonary disease (COPD) has been well documented, with airway smooth muscle cell proliferation and migration playing a role in the remodeling process. Here, we aimed to verify the effects of fine particulate matter (PM2.5) on human bronchial smooth muscle cell (HBSMC) migration and to explore the underlying signaling pathways. HBSMC apoptosis, proliferation and migration were measured using flow cytometry, cell counting and transwell migration assays, respectively. The role of the hedgehog pathway in cell migration was assessed by western blotting to measure the expression of Sonic hedgehog (Shh), Gli1 and Snail. Furthermore, siRNA was used to knock down Gli1 or Snail expression. PM2.5 induced HBSMC apoptosis in a dose-dependent manner, although certain concentrations of PM2.5 did not induce HBSMC proliferation or apoptosis. Interestingly, cell migration was stimulated by PM2.5 doses far below those that induced apoptosis. Additional experiments revealed that these PM2.5 doses enhanced the expression of Shh, Gli1 and Snail in HBSMCs. Furthermore, PM2.5-induced cell migration and protein expression were enhanced by recombinant Shh and attenuated by cyclopamine. Similar results were obtained by knocking down Gli1 or Snail. These findings suggest that PM2.5, which may exert its effects through the Shh signaling pathway, is necessary for the migration of HBSMCs. These data define a novel role for PM2.5 in airway remodeling in COPD.

HPIminer: A text mining system for building and visualizing human protein interaction networks and pathways.

PubMed

Subramani, Suresh; Kalpana, Raja; Monickaraj, Pankaj Moses; Natarajan, Jeyakumar

2015-04-01

The knowledge on protein-protein interactions (PPI) and their related pathways are equally important to understand the biological functions of the living cell. Such information on human proteins is highly desirable to understand the mechanism of several diseases such as cancer, diabetes, and Alzheimer's disease. Because much of that information is buried in biomedical literature, an automated text mining system for visualizing human PPI and pathways is highly desirable. In this paper, we present HPIminer, a text mining system for visualizing human protein interactions and pathways from biomedical literature. HPIminer extracts human PPI information and PPI pairs from biomedical literature, and visualize their associated interactions, networks and pathways using two curated databases HPRD and KEGG. To our knowledge, HPIminer is the first system to build interaction networks from literature as well as curated databases. Further, the new interactions mined only from literature and not reported earlier in databases are highlighted as new. A comparative study with other similar tools shows that the resultant network is more informative and provides additional information on interacting proteins and their associated networks. Copyright © 2015 Elsevier Inc. All rights reserved.

Computational Framework for Analysis of Prey–Prey Associations in Interaction Proteomics Identifies Novel Human Protein–Protein Interactions and Networks

PubMed Central

Saha, Sudipto; Dazard, Jean-Eudes; Xu, Hua; Ewing, Rob M.

2013-01-01

Large-scale protein–protein interaction data sets have been generated for several species including yeast and human and have enabled the identification, quantification, and prediction of cellular molecular networks. Affinity purification-mass spectrometry (AP-MS) is the preeminent methodology for large-scale analysis of protein complexes, performed by immunopurifying a specific “bait” protein and its associated “prey” proteins. The analysis and interpretation of AP-MS data sets is, however, not straightforward. In addition, although yeast AP-MS data sets are relatively comprehensive, current human AP-MS data sets only sparsely cover the human interactome. Here we develop a framework for analysis of AP-MS data sets that addresses the issues of noise, missing data, and sparsity of coverage in the context of a current, real world human AP-MS data set. Our goal is to extend and increase the density of the known human interactome by integrating bait–prey and cocomplexed preys (prey–prey associations) into networks. Our framework incorporates a score for each identified protein, as well as elements of signal processing to improve the confidence of identified protein–protein interactions. We identify many protein networks enriched in known biological processes and functions. In addition, we show that integrated bait–prey and prey–prey interactions can be used to refine network topology and extend known protein networks. PMID:22845868

Yeast Gup1(2) Proteins Are Homologues of the Hedgehog Morphogens Acyltransferases HHAT(L): Facts and Implications

PubMed Central

Lucas, Cândida; Ferreira, Célia; Cazzanelli, Giulia; Franco-Duarte, Ricardo; Tulha, Joana

2016-01-01

In multiple tissues, the Hedgehog secreted morphogen activates in the receiving cells a pathway involved in cell fate, proliferation and differentiation in the receiving cells. This pathway is particularly important during embryogenesis. The protein HHAT (Hedgehog O-acyltransferase) modifies Hh morphogens prior to their secretion, while HHATL (Hh O-acyltransferase-like) negatively regulates the pathway. HHAT and HHATL are homologous to Saccharomyces cerevisiae Gup2 and Gup1, respectively. In yeast, Gup1 is associated with a high number and diversity of biological functions, namely polarity establishment, secretory/endocytic pathway functionality, vacuole morphology and wall and membrane composition, structure and maintenance. Phenotypes underlying death, morphogenesis and differentiation are also included. Paracrine signalling, like the one promoted by the Hh pathway, has not been shown to occur in microbial communities, despite the fact that large aggregates of cells like biofilms or colonies behave as proto-tissues. Instead, these have been suggested to sense the population density through the secretion of quorum-sensing chemicals. This review focuses on Gup1/HHATL and Gup2/HHAT proteins. We review the functions and physiology associated with these proteins in yeasts and higher eukaryotes. We suggest standardisation of the presently chaotic Gup-related nomenclature, which includes KIAA117, c3orf3, RASP, Skinny, Sightless and Central Missing, in order to avoid the disclosure of otherwise unnoticed information. PMID:29615596

Predicting Human Protein Subcellular Locations by the Ensemble of Multiple Predictors via Protein-Protein Interaction Network with Edge Clustering Coefficients

PubMed Central

Du, Pufeng; Wang, Lusheng

2014-01-01

One of the fundamental tasks in biology is to identify the functions of all proteins to reveal the primary machinery of a cell. Knowledge of the subcellular locations of proteins will provide key hints to reveal their functions and to understand the intricate pathways that regulate biological processes at the cellular level. Protein subcellular location prediction has been extensively studied in the past two decades. A lot of methods have been developed based on protein primary sequences as well as protein-protein interaction network. In this paper, we propose to use the protein-protein interaction network as an infrastructure to integrate existing sequence based predictors. When predicting the subcellular locations of a given protein, not only the protein itself, but also all its interacting partners were considered. Unlike existing methods, our method requires neither the comprehensive knowledge of the protein-protein interaction network nor the experimentally annotated subcellular locations of most proteins in the protein-protein interaction network. Besides, our method can be used as a framework to integrate multiple predictors. Our method achieved 56% on human proteome in absolute-true rate, which is higher than the state-of-the-art methods. PMID:24466278

Evolutionary analyses of hedgehog and Hoxd-10 genes in fish species closely related to the zebrafish

PubMed Central

Zardoya, Rafael; Abouheif, Ehab; Meyer, Axel

1996-01-01

The study of development has relied primarily on the isolation of mutations in genes with specific functions in development and on the comparison of their expression patterns in normal and mutant phenotypes. Comparative evolutionary analyses can complement these approaches. Phylogenetic analyses of Sonic hedgehog (Shh) and Hoxd-10 genes from 18 cyprinid fish species closely related to the zebrafish provide novel insights into the functional constraints acting on Shh. Our results confirm and extend those gained from expression and crystalline structure analyses of this gene. Unexpectedly, exon 1 of Shh is found to be almost invariant even in third codon positions among these morphologically divergent species suggesting that this exon encodes for a functionally important domain of the hedgehog protein. This is surprising because the main functional domain of Shh had been thought to be that encoded by exon 2. Comparisons of Shh and Hoxd-10 gene sequences and of resulting gene trees document higher evolutionary constraints on the former than on the latter. This might be indicative of more general evolutionary patterns in networks of developmental regulatory genes interacting in a hierarchical fashion. The presence of four members of the hedgehog gene family in cyprinid fishes was documented and their homologies to known hedgehog genes in other vertebrates were established. PMID:8917540

Evolutionary analyses of hedgehog and Hoxd-10 genes in fish species closely related to the zebrafish.

PubMed

Zardoya, R; Abouheif, E; Meyer, A

1996-11-12

The study of development has relied primarily on the isolation of mutations in genes with specific functions in development and on the comparison of their expression patterns in normal and mutant phenotypes. Comparative evolutionary analyses can complement these approaches. Phylogenetic analyses of Sonic hedgehog (Shh) and Hoxd-10 genes from 18 cyprinid fish species closely related to the zebrafish provide novel insights into the functional constraints acting on Shh. Our results confirm and extend those gained from expression and crystalline structure analyses of this gene. Unexpectedly, exon 1 of Shh is found to be almost invariant even in third codon positions among these morphologically divergent species suggesting that this exon encodes for a functionally important domain of the hedgehog protein. This is surprising because the main functional domain of Shh had been thought to be that encoded by exon 2. Comparisons of Shh and Hoxd-10 gene sequences and of resulting gene trees document higher evolutionary constraints on the former than on the latter. This might be indicative of more general evolutionary patterns in networks of developmental regulatory genes interacting in a hierarchical fashion. The presence of four members of the hedgehog gene family in cyprinid fishes was documented and their homologies to known hedgehog genes in other vertebrates were established.

Interaction between human BAP31 and respiratory syncytial virus small hydrophobic (SH) protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Yan; Jain, Neeraj; Limpanawat, Suweeraya

2015-08-15

The small hydrophobic (SH) protein is a short channel-forming polypeptide encoded by the human respiratory syncytial virus (hRSV). Deletion of SH protein leads to the viral attenuation in mice and primates, and delayed apoptosis in infected cells. We have used a membrane-based yeast two-hybrid system (MbY2H) and a library from human lung cDNA to detect proteins that bind SH protein. This led to the identification of a membrane protein, B-cell associated protein 31 (BAP31). Transfected SH protein co-localizes with transfected BAP31 in cells, and pulls down endogenous BAP31. Titration of purified C-terminal endodomain of BAP31 against isotopically labeled SH proteinmore » in detergent micelles suggests direct interaction between the two proteins. Given the key role of BAP31 in protein trafficking and its critical involvement in pro- and anti-apoptotic pathways, this novel interaction may constitute a potential drug target. - Highlights: • A yeast two-hybrid system (MbY2H) detected BAP31 as a binder of RSV SH protein. • Transfected SH and BAP31 co-localize in lung epithelial cells. • Endogenous BAP31 is pulled down by RSV SH protein. • BAP31 endodomain interacts with the N-terminal α-helix of SH protein in micelles. • This interaction is proposed to be a potential drug target.« less

Comprehensive prediction of drug-protein interactions and side effects for the human proteome

PubMed Central

Zhou, Hongyi; Gao, Mu; Skolnick, Jeffrey

2015-01-01

Identifying unexpected drug-protein interactions is crucial for drug repurposing. We develop a comprehensive proteome scale approach that predicts human protein targets and side effects of drugs. For drug-protein interaction prediction, FINDSITEcomb, whose average precision is ~30% and recall ~27%, is employed. For side effect prediction, a new method is developed with a precision of ~57% and a recall of ~24%. Our predictions show that drugs are quite promiscuous, with the average (median) number of human targets per drug of 329 (38), while a given protein interacts with 57 drugs. The result implies that drug side effects are inevitable and existing drugs may be useful for repurposing, with only ~1,000 human proteins likely causing serious side effects. A killing index derived from serious side effects has a strong correlation with FDA approved drugs being withdrawn. Therefore, it provides a pre-filter for new drug development. The methodology is free to the academic community on the DR. PRODIS (DRugome, PROteome, and DISeasome) webserver at http://cssb.biology.gatech.edu/dr.prodis/. DR. PRODIS provides protein targets of drugs, drugs for a given protein target, associated diseases and side effects of drugs, as well as an interface for the virtual target screening of new compounds. PMID:26057345

Epigenetic targeting of Hedgehog pathway transcriptional output through BET bromodomain inhibition

PubMed Central

Tang, Yujie; Gholamin, Sharareh; Schubert, Simone; Willardson, Minde I.; Lee, Alex; Bandopadhayay, Pratiti; Bergthold, Guillame; Masoud, Sabran; Nguyen, Brian; Vue, Nujsaubnusi; Balansay, Brianna; Yu, Furong; Oh, Sekyung; Woo, Pamelyn; Chen, Spenser; Ponnuswami, Anitha; Monje, Michelle; Atwood, Scott X.; Whitson, Ramon J.; Mitra, Siddhartha; Cheshier, Samuel H.; Qi, Jun; Beroukhim, Rameen; Tang, Jean Y.; Wechsler-Reya, Rob; Oro, Anthony E.; Link, Brian A.; Bradner, James E.; Cho, Yoon-Jae

2014-01-01

Hedgehog signaling drives oncogenesis in several cancers and strategies targeting this pathway have been developed, most notably through inhibition of Smoothened. However, resistance to Smoothened inhibitors occurs via genetic changes of Smoothened or other downstream Hedgehog components. Here, we overcome these resistance mechanisms by modulating GLI transcription via inhibition of BET bromodomain proteins. We show the BET bromodomain protein, BRD4, regulates GLI transcription downstream of SMO and SUFU and chromatin immunoprecipitation studies reveal BRD4 directly occupies GLI1 and GLI2 promoters, with a substantial decrease in engagement of these sites upon treatment with JQ1, a small molecule inhibitor targeting BRD4. Globally, genes associated with medulloblastoma-specific GLI1 binding sites are downregulated in response to JQ1 treatment, supporting direct regulation of GLI activity by BRD4. Notably, patient- and GEMM-derived Hedgehog-driven tumors (basal cell carcinoma, medulloblastoma and atypical teratoid/rhabdoid tumor) respond to JQ1 even when harboring genetic lesions rendering them resistant to Smoothened antagonists. PMID:24973920

Phosphoproteome analysis reveals a critical role for hedgehog signalling in osteoblast morphological transitions.

PubMed

Marumoto, Ariane; Milani, Renato; da Silva, Rodrigo A; da Costa Fernandes, Célio Junior; Granjeiro, José Mauro; Ferreira, Carmen V; Peppelenbosch, Maikel P; Zambuzzi, Willian F

2017-10-01

The reciprocal and adaptive interactions between cells and substrates governing morphological transitions in the osteoblast compartment remain largely obscure. Here we show that osteoblast cultured in basement membrane matrix (Matrigel™) exhibits significant morphological changes after ten days of culture, and we decided to exploit this situation to investigate the molecular mechanisms responsible for guiding osteoblast morphological transitions. As almost all aspects of cellular physiology are under control of kinases, we generated more or less comprehensive cellular kinome profiles employing PepChip peptide arrays that contain over 1000 consensus substrates of kinase peptide. The results obtained were used to construct interactomes, and these revealed an important role for FoxO in mediating morphological changes of osteoblast, which was validated by Western blot technology when FoxO was significantly up-expressed in response to Matrigel™. As FoxO is a critical protein in canonical hedgehog signalling, we decided to explore the possible involvement of hedgehog signalling during osteoblast morphological changes. It appeared that osteoblast culture in Matrigel™ stimulates release of a substantial amounts Shh while concomitantly inducing upregulation of the expression of the bona fide hedgehog target genes Gli-1 and Patched. Functional confirmation of the relevance of these results for osteoblast morphological transitions came from experiments in which Shh hedgehog signalling was inhibited using the well-established pathway inhibitor cyclopamine (Cyc). In the presence of Cyc, culture of osteoblasts in Matrigel™ is not capable of inducing morphological changes but appears to provoke a proliferative response as evident from the upregulation of Cyclin D3 and cdk4. The most straightforward interpretation of our results is that hedgehog signalling is both necessary and sufficient for membrane matrix-based morphological transitions. Copyright © 2017 Elsevier Inc

Position Matters: Network Centrality Considerably Impacts Rates of Protein Evolution in the Human Protein-Protein Interaction Network.

PubMed

Alvarez-Ponce, David; Feyertag, Felix; Chakraborty, Sandip

2017-06-01

The proteins of any organism evolve at disparate rates. A long list of factors affecting rates of protein evolution have been identified. However, the relative importance of each factor in determining rates of protein evolution remains unresolved. The prevailing view is that evolutionary rates are dominantly determined by gene expression, and that other factors such as network centrality have only a marginal effect, if any. However, this view is largely based on analyses in yeasts, and accurately measuring the importance of the determinants of rates of protein evolution is complicated by the fact that the different factors are often correlated with each other, and by the relatively poor quality of available functional genomics data sets. Here, we use correlation, partial correlation and principal component regression analyses to measure the contributions of several factors to the variability of the rates of evolution of human proteins. For this purpose, we analyzed the entire human protein-protein interaction data set and the human signal transduction network-a network data set of exceptionally high quality, obtained by manual curation, which is expected to be virtually free from false positives. In contrast with the prevailing view, we observe that network centrality (measured as the number of physical and nonphysical interactions, betweenness, and closeness) has a considerable impact on rates of protein evolution. Surprisingly, the impact of centrality on rates of protein evolution seems to be comparable, or even superior according to some analyses, to that of gene expression. Our observations seem to be independent of potentially confounding factors and from the limitations (biases and errors) of interactomic data sets. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Maintenance of Taste Organs Is Strictly Dependent on Epithelial Hedgehog/GLI Signaling.

PubMed

Ermilov, Alexandre N; Kumari, Archana; Li, Libo; Joiner, Ariell M; Grachtchouk, Marina A; Allen, Benjamin L; Dlugosz, Andrzej A; Mistretta, Charlotte M

2016-11-01

For homeostasis, lingual taste papilla organs require regulation of epithelial cell survival and renewal, with sustained innervation and stromal interactions. To investigate a role for Hedgehog/GLI signaling in adult taste organs we used a panel of conditional mouse models to manipulate GLI activity within epithelial cells of the fungiform and circumvallate papillae. Hedgehog signaling suppression rapidly led to taste bud loss, papilla disruption, and decreased proliferation in domains of papilla epithelium that contribute to taste cells. Hedgehog responding cells were eliminated from the epithelium but retained in the papilla stromal core. Despite papilla disruption and loss of taste buds that are a major source of Hedgehog ligand, innervation to taste papillae was maintained, and not misdirected, even after prolonged GLI blockade. Further, vimentin-positive fibroblasts remained in the papilla core. However, retained innervation and stromal cells were not sufficient to maintain taste bud cells in the context of compromised epithelial Hedgehog signaling. Importantly taste organ disruption after GLI blockade was reversible in papillae that retained some taste bud cell remnants where reactivation of Hedgehog signaling led to regeneration of papilla epithelium and taste buds. Therefore, taste bud progenitors were either retained during epithelial GLI blockade or readily repopulated during recovery, and were poised to regenerate taste buds once Hedgehog signaling was restored, with innervation and papilla connective tissue elements in place. Our data argue that Hedgehog signaling is essential for adult tongue tissue maintenance and that taste papilla epithelial cells represent the key targets for physiologic Hedgehog-dependent regulation of taste organ homeostasis. Because disruption of GLI transcriptional activity in taste papilla epithelium is sufficient to drive taste organ loss, similar to pharmacologic Hedgehog pathway inhibition, the findings suggest that taste

Maintenance of Taste Organs Is Strictly Dependent on Epithelial Hedgehog/GLI Signaling

PubMed Central

Mistretta, Charlotte M.

2016-01-01

For homeostasis, lingual taste papilla organs require regulation of epithelial cell survival and renewal, with sustained innervation and stromal interactions. To investigate a role for Hedgehog/GLI signaling in adult taste organs we used a panel of conditional mouse models to manipulate GLI activity within epithelial cells of the fungiform and circumvallate papillae. Hedgehog signaling suppression rapidly led to taste bud loss, papilla disruption, and decreased proliferation in domains of papilla epithelium that contribute to taste cells. Hedgehog responding cells were eliminated from the epithelium but retained in the papilla stromal core. Despite papilla disruption and loss of taste buds that are a major source of Hedgehog ligand, innervation to taste papillae was maintained, and not misdirected, even after prolonged GLI blockade. Further, vimentin-positive fibroblasts remained in the papilla core. However, retained innervation and stromal cells were not sufficient to maintain taste bud cells in the context of compromised epithelial Hedgehog signaling. Importantly taste organ disruption after GLI blockade was reversible in papillae that retained some taste bud cell remnants where reactivation of Hedgehog signaling led to regeneration of papilla epithelium and taste buds. Therefore, taste bud progenitors were either retained during epithelial GLI blockade or readily repopulated during recovery, and were poised to regenerate taste buds once Hedgehog signaling was restored, with innervation and papilla connective tissue elements in place. Our data argue that Hedgehog signaling is essential for adult tongue tissue maintenance and that taste papilla epithelial cells represent the key targets for physiologic Hedgehog-dependent regulation of taste organ homeostasis. Because disruption of GLI transcriptional activity in taste papilla epithelium is sufficient to drive taste organ loss, similar to pharmacologic Hedgehog pathway inhibition, the findings suggest that taste

Basal cell carcinoma pathogenesis and therapy involving hedgehog signaling and beyond.

PubMed

Bakshi, Anshika; Chaudhary, Sandeep C; Rana, Mehtab; Elmets, Craig A; Athar, Mohammad

2017-12-01

Basal cell carcinoma (BCC) of the skin is driven by aberrant hedgehog signaling. Thus blocking this signaling pathway by small molecules such as vismodegib inhibits tumor growth. Primary cilium in the epidermal cells plays an integral role in the processing of hedgehog signaling-related proteins. Recent genomic studies point to the involvement of additional genetic mutations that might be associated with the development of BCCs, suggesting significance of other signaling pathways, such as WNT, NOTCH, mTOR, and Hippo, aside from hedgehog in the pathogenesis of this human neoplasm. Some of these pathways could be regulated by noncoding microRNA. Altered microRNA expression profile is recognized with the progression of these lesions. Stopping treatment with Smoothened (SMO) inhibitors often leads to tumor reoccurrence in the patients with basal cell nevus syndrome, who develop 10-100 of BCCs. In addition, the initial effectiveness of these SMO inhibitors is impaired due to the onset of mutations in the drug-binding domain of SMO. These data point to a need to develop strategies to overcome tumor recurrence and resistance and to enhance efficacy by developing novel single agent-based or multiple agents-based combinatorial approaches. Immunotherapy and photodynamic therapy could be additional successful approaches particularly if developed in combination with chemotherapy for inoperable and metastatic BCCs. © 2017 Wiley Periodicals, Inc.

Basal cell carcinoma pathogenesis and therapy involving hedgehog signaling and beyond

PubMed Central

Bakshi, Anshika; Chaudhary, Sandeep C.; Rana, Mehtab; Elmets, Craig A.; Athar, Mohammad

2018-01-01

Basal cell carcinoma (BCC) of the skin is driven by aberrant hedgehog signaling. Thus blocking this signaling pathway by small molecules such as vismodegib inhibits tumor growth. Primary cilium in the epidermal cells plays an integral role in the processing of hedgehog signaling-related proteins. Recent genomic studies point to the involvement of additional genetic mutations that might be associated with the development of BCCs, suggesting significance of other signaling pathways, such as WNT, NOTCH, mTOR, and Hippo, aside from hedgehog in the pathogenesis of this human neoplasm. Some of these pathways could be regulated by noncoding microRNA. Altered microRNA expression profile is recognized with the progression of these lesions. Stopping treatment with Smoothened (SMO) inhibitors often leads to tumor reoccurrence in the patients with basal cell nevus syndrome, who develop 10–100 of BCCs. In addition, the initial effectiveness of these SMO inhibitors is impaired due to the onset of mutations in the drug-binding domain of SMO. These data point to a need to develop strategies to overcome tumor recurrence and resistance and to enhance efficacy by developing novel single agent-based or multiple agents-based combinatorial approaches. Immunotherapy and photodynamic therapy could be additional successful approaches particularly if developed in combination with chemotherapy for inoperable and metastatic BCCs. PMID:28574612

Hedgehog induction of murine vasculogenesis is mediated by Foxf1 and Bmp4.

PubMed

Astorga, Jeanette; Carlsson, Peter

2007-10-01

The first vasculature of the developing vertebrate embryo forms by assembly of endothelial cells into simple tubes from clusters of mesodermal angioblasts. Maturation of this vasculature involves remodeling, pruning and investment with mural cells. Hedgehog proteins are part of the instructive endodermal signal that triggers the assembly of the first primitive vessels in the mesoderm. We used a combination of genetic and in vitro culture methods to investigate the role of hedgehogs and their targets in murine extraembryonic vasculogenesis. We show that Bmps, in particular Bmp4, are crucial for vascular tube formation, that Bmp4 expression in extraembryonic tissues requires the forkhead transcription factor Foxf1 and that the role of hedgehog proteins in this process is to activate Foxf1 expression in the mesoderm. We show in the allantois that genetic disruption of hedgehog signaling (Smo(-/-)) has no effect on Foxf1 expression, and neither Bmp4 expression nor vasculogenesis are disturbed. By contrast, targeted inactivation of Foxf1 leads to loss of allantoic Bmp4 and vasculature. In vitro, the avascular Foxf1(-/-) phenotype can be rescued by exogenous Bmp4, and vasculogenesis in wild-type tissue can be blocked by the Bmp antagonist noggin. Hedgehogs are required for activation of Foxf1, Bmp4 expression and vasculogenesis in the yolk sac. However, vasculogenesis in Smo(-/-) yolk sacs can be rescued by exogenous Bmp4, consistent with the notion that the role of hedgehog signaling in primary vascular tube formation is as an activator of Bmp4, via Foxf1.

Decoding the phosphorylation code in Hedgehog signal transduction

PubMed Central

Chen, Yongbin; Jiang, Jin

2013-01-01

Hedgehog (Hh) signaling plays pivotal roles in embryonic development and adult tissue homeostasis, and its deregulation leads to numerous human disorders including cancer. Binding of Hh to Patched (Ptc), a twelve-transmembrane protein, alleviates its inhibition of Smoothened (Smo), a seven-transmembrane protein related to G-protein-coupled receptors (GPCRs), leading to Smo phosphorylation and activation. Smo acts through intracellular signaling complexes to convert the latent transcription factor Cubitus interruptus (Ci)/Gli from a truncated repressor to a full-length activator, leading to derepression/activation of Hh target genes. Increasing evidence suggests that phosphorylation participates in almost every step in the signal relay from Smo to Ci/Gli, and that differential phosphorylation of several key pathway components may be crucial for translating the Hh morphogen gradient into graded pathway activities. In this review, we focus on the multifaceted roles that phosphorylation plays in Hh signal transduction, and discuss the conservation and difference between Drosophila and mammalian Hh signaling mechanisms. PMID:23337587

Interaction of Proteins Identified in Human Thyroid Cells

PubMed Central

Pietsch, Jessica; Riwaldt, Stefan; Bauer, Johann; Sickmann, Albert; Weber, Gerhard; Grosse, Jirka; Infanger, Manfred; Eilles, Christoph; Grimm, Daniela

2013-01-01

Influence of gravity forces on the regulation of protein expression by healthy and malignant thyroid cells was studied with the aim to identify protein interactions. Western blot analyses of a limited number of proteins suggested a time-dependent regulation of protein expression by simulated microgravity. After applying free flow isoelectric focusing and mass spectrometry to search for differently expressed proteins by thyroid cells exposed to simulated microgravity for three days, a considerable number of candidates for gravi-sensitive proteins were detected. In order to show how proteins sensitive to microgravity could directly influence other proteins, we investigated all polypeptide chains identified with Mascot scores above 100, looking for groups of interacting proteins. Hence, UniProtKB entry numbers of all detected proteins were entered into the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and processed. The program indicated that we had detected various groups of interacting proteins in each of the three cell lines studied. The major groups of interacting proteins play a role in pathways of carbohydrate and protein metabolism, regulation of cell growth and cell membrane structuring. Analyzing these groups, networks of interaction could be established which show how a punctual influence of simulated microgravity may propagate via various members of interaction chains. PMID:23303277

Computational prediction of host-pathogen protein-protein interactions.

PubMed

Dyer, Matthew D; Murali, T M; Sobral, Bruno W

2007-07-01

Infectious diseases such as malaria result in millions of deaths each year. An important aspect of any host-pathogen system is the mechanism by which a pathogen can infect its host. One method of infection is via protein-protein interactions (PPIs) where pathogen proteins target host proteins. Developing computational methods that identify which PPIs enable a pathogen to infect a host has great implications in identifying potential targets for therapeutics. We present a method that integrates known intra-species PPIs with protein-domain profiles to predict PPIs between host and pathogen proteins. Given a set of intra-species PPIs, we identify the functional domains in each of the interacting proteins. For every pair of functional domains, we use Bayesian statistics to assess the probability that two proteins with that pair of domains will interact. We apply our method to the Homo sapiens-Plasmodium falciparum host-pathogen system. Our system predicts 516 PPIs between proteins from these two organisms. We show that pairs of human proteins we predict to interact with the same Plasmodium protein are close to each other in the human PPI network and that Plasmodium pairs predicted to interact with same human protein are co-expressed in DNA microarray datasets measured during various stages of the Plasmodium life cycle. Finally, we identify functionally enriched sub-networks spanned by the predicted interactions and discuss the plausibility of our predictions. Supplementary data are available at http://staff.vbi.vt.edu/dyermd/publications/dyer2007a.html. Supplementary data are available at Bioinformatics online.

Indian hedgehog contributes to human cartilage endplate degeneration.

PubMed

Wang, Shaowei; Yang, Kun; Chen, Shuai; Wang, Jiying; Du, Guoqing; Fan, Shunwu; Wei, Lei

2015-08-01

To determine the role of Indian hedgehog (Ihh) signaling in human cartilage endplate (CEP) degeneration. CEP-degenerated tissues from patients with Modic I or II changes (n = 9 and 45, respectively) and normal tissues from vertebral burst fracture patients (n = 17) were collected. Specimens were either cut into slices for organ culture ex vivo or digested to isolate chondrocytes for cell culture in vitro. Ihh expression and the effect of Ihh on cartilage degeneration were determined by investigating degeneration markers in this study. Ihh expression and cartilage degeneration markers significantly increased in the Modic I and II groups. The expression of cartilage degeneration markers was positively correlated with degeneration severity. Gain-of-function for Ihh promoted expression of cartilage degeneration markers in vitro, while loss-of-function for Ihh inhibited their expression both in vitro and ex vivo. These findings demonstrated that Ihh promotes CEP degeneration. Blocking Ihh pathway has potential clinical usage for attenuating CEP degeneration.

PICKLE 2.0: A human protein-protein interaction meta-database employing data integration via genetic information ontology

PubMed Central

Gioutlakis, Aris; Klapa, Maria I.

2017-01-01

It has been acknowledged that source databases recording experimentally supported human protein-protein interactions (PPIs) exhibit limited overlap. Thus, the reconstruction of a comprehensive PPI network requires appropriate integration of multiple heterogeneous primary datasets, presenting the PPIs at various genetic reference levels. Existing PPI meta-databases perform integration via normalization; namely, PPIs are merged after converted to a certain target level. Hence, the node set of the integrated network depends each time on the number and type of the combined datasets. Moreover, the irreversible a priori normalization process hinders the identification of normalization artifacts in the integrated network, which originate from the nonlinearity characterizing the genetic information flow. PICKLE (Protein InteraCtion KnowLedgebasE) 2.0 implements a new architecture for this recently introduced human PPI meta-database. Its main novel feature over the existing meta-databases is its approach to primary PPI dataset integration via genetic information ontology. Building upon the PICKLE principles of using the reviewed human complete proteome (RHCP) of UniProtKB/Swiss-Prot as the reference protein interactor set, and filtering out protein interactions with low probability of being direct based on the available evidence, PICKLE 2.0 first assembles the RHCP genetic information ontology network by connecting the corresponding genes, nucleotide sequences (mRNAs) and proteins (UniProt entries) and then integrates PPI datasets by superimposing them on the ontology network without any a priori transformations. Importantly, this process allows the resulting heterogeneous integrated network to be reversibly normalized to any level of genetic reference without loss of the original information, the latter being used for identification of normalization biases, and enables the appraisal of potential false positive interactions through PPI source database cross-checking. The

The Orphan G Protein-coupled Receptor Gpr175 (Tpra40) Enhances Hedgehog Signaling by Modulating cAMP Levels.

PubMed

Singh, Jaskirat; Wen, Xiaohui; Scales, Suzie J

2015-12-04

The Hedgehog (Hh) signaling pathway plays an essential role in vertebrate embryonic tissue patterning of many developing organs. Signaling occurs predominantly in primary cilia and is initiated by the entry of the G protein-coupled receptor (GPCR)-like protein Smoothened into cilia and culminates in gene transcription via the Gli family of transcription factors upon their nuclear entry. Here we identify an orphan GPCR, Gpr175 (also known as Tpra1 or Tpra40: transmembrane protein, adipocyte associated 1 or of 40 kDa), which also localizes to primary cilia upon Hh stimulation and positively regulates Hh signaling. Interaction experiments place Gpr175 at the level of PKA and upstream of the Gαi component of heterotrimeric G proteins, which itself localizes to cilia and can modulate Hh signaling. Gpr175 or Gαi1 depletion leads to increases in cellular cAMP levels and in Gli3 processing into its repressor form. Thus we propose that Gpr175 coupled to Gαi1 normally functions to inhibit the production of cAMP by adenylyl cyclase upon Hh stimulation, thus maximizing signaling by turning off PKA activity and hence Gli3 repressor formation. Taken together our data suggest that Gpr175 is a novel positive regulator of the Hh signaling pathway. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Single-nucleotide variants in two Hedgehog genes, SHH and HHIP, as genetic cause of combined pituitary hormone deficiency.

PubMed

Gorbenko del Blanco, Darya; de Graaff, Laura C G; Visser, Theo J; Hokken-Koelega, Anita C S

2013-03-01

Combined pituitary hormone deficiency (CPHD) is characterized by deficiencies of two or more anterior pituitary hormones. Its genetic cause is unknown in the majority of cases. The Hedgehog (Hh) signalling pathway has been implicated in disorders associated with pituitary development. Mutations in Sonic Hedgehog (SHH) have been described in patients with holoprosencephaly (with or without pituitary involvement). Hedgehog interacting protein (HHIP) has been associated with variations in adult height in genome wide association studies. We investigated whether mutations in these two genes of the Hh pathway, SHH and HHIP, could result in 'idiopathic' CPHD. We directly sequenced the coding regions and exon - intron boundaries of SHH and HHIP in 93 CPHD patients of the Dutch HYPOPIT study in whom mutations in the classical CPHD genes PROP1, POU1F1, HESX1, LHX3 and LHX4 had been ruled out. We compared the expression of Hh genes in Hep3B transfected cells between wild-type proteins and mutants. We identified three single-nucleotide variants (p.Ala226Thr, c.1078C>T and c.*8G>T) in SHH. The function of the latter was severely affected in our in vitro assay. In HHIP, we detected a new activating variant c.-1G>C, which increases HHIP's inhibiting function on the Hh pathway. Our results suggest involvement of the Hedgehog pathway in CPHD. We suggest that both SHH and HHIP are investigated as a second screening in CPHD, after mutations in the classical CPHD genes have been ruled out. © 2012 Blackwell Publishing Ltd.

Protein-bound Polysaccharide-K Inhibits Hedgehog Signaling Through Down-regulation of MAML3 and RBPJ Transcription Under Hypoxia, Suppressing the Malignant Phenotype in Pancreatic Cancer.

PubMed

Yamasaki, Akio; Onishi, Hideya; Imaizumi, Akira; Kawamoto, Makoto; Fujimura, Akiko; Oyama, Yasuhiro; Katano, Mitsuo

2016-08-01

Hedgehog signaling is activated in pancreatic cancer and could be a therapeutic target. We previously demonstrated that recombination signal binding protein for immunoglobulin-kappa-J region (RBPJ) and mastermind-like 3 (MAML3) contribute to the hypoxia-induced up-regulation of Smoothened (SMO) transcription. We have also shown that protein-bound polysaccharide-K (PSK) could be effective for refractory pancreatic cancer that down-regulates SMO transcription under hypoxia. In this study, we evaluated whether the anticancer mechanism of PSK involves inhibiting RBPJ and MAML3 expression under hypoxia. PSK reduced SMO, MAML3 and RBPJ expression in pancreatic cancer cells under hypoxia. PSK also blocked RBPJ-induced invasiveness under hypoxia by inhibiting matrix metalloproteinase expression. Lastly, we showed that PSK attenuated RBPJ-induced proliferation both in vitro and in vivo. These results suggest that PSK suppresses Hedgehog signaling through down-regulation of MAML3 and RBPJ transcription under hypoxia, inhibiting the induction of a malignant phenotype in pancreatic cancer. Our results may lead to development of new treatments for refractory pancreatic cancer using PSK as a Hedgehog inhibitor. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Outfoxing the Hedgehog

ERIC Educational Resources Information Center

Barbieri, Richard

2011-01-01

Jim Collins's "Good to Great" has attained near-scriptural status in organizations, including nonprofits, which Collins says constitute a third of his readers. The pivot point in "Good to Great" is the Hedgehog Concept. The "Hedgehog Concept" (HC), this author claims, is dangerous for schools because it distorts the nature of education. As Collins…

LocFuse: human protein-protein interaction prediction via classifier fusion using protein localization information.

PubMed

Zahiri, Javad; Mohammad-Noori, Morteza; Ebrahimpour, Reza; Saadat, Samaneh; Bozorgmehr, Joseph H; Goldberg, Tatyana; Masoudi-Nejad, Ali

2014-12-01

Protein-protein interaction (PPI) detection is one of the central goals of functional genomics and systems biology. Knowledge about the nature of PPIs can help fill the widening gap between sequence information and functional annotations. Although experimental methods have produced valuable PPI data, they also suffer from significant limitations. Computational PPI prediction methods have attracted tremendous attentions. Despite considerable efforts, PPI prediction is still in its infancy in complex multicellular organisms such as humans. Here, we propose a novel ensemble learning method, LocFuse, which is useful in human PPI prediction. This method uses eight different genomic and proteomic features along with four types of different classifiers. The prediction performance of this classifier selection method was found to be considerably better than methods employed hitherto. This confirms the complex nature of the PPI prediction problem and also the necessity of using biological information for classifier fusion. The LocFuse is available at: http://lbb.ut.ac.ir/Download/LBBsoft/LocFuse. The results revealed that if we divide proteome space according to the cellular localization of proteins, then the utility of some classifiers in PPI prediction can be improved. Therefore, to predict the interaction for any given protein pair, we can select the most accurate classifier with regard to the cellular localization information. Based on the results, we can say that the importance of different features for PPI prediction varies between differently localized proteins; however in general, our novel features, which were extracted from position-specific scoring matrices (PSSMs), are the most important ones and the Random Forest (RF) classifier performs best in most cases. LocFuse was developed with a user-friendly graphic interface and it is freely available for Linux, Mac OSX and MS Windows operating systems. Copyright © 2014 Elsevier Inc. All rights reserved.

A protein isolated from human oviductal tissue in vitro secretion, identified as human lactoferrin, interacts with spermatozoa and oocytes and modulates gamete interaction.

PubMed

Zumoffen, C M; Gil, R; Caille, A M; Morente, C; Munuce, M J; Ghersevich, S A

2013-05-01

Is lactoferrin (LF) (detected in oviductal secretion) able to bind to oocytes and sperm and modulate gamete interaction? LF binds to zona pellucida (ZP) and spermatozoa (depending upon the capacitation stage and acrosome status) and inhibits gamete interaction in vitro. Proteins from human oviductal tissue secretion modulate gamete interaction and parameters of sperm function in vitro and some of them bind to sperm, but they remain to be isolated and identified. Proteins were isolated from human oviductal tissue secretion using their sperm membrane binding ability. One of the isolated proteins was identified as human LF and immunolocalized in tubal tissues. LF expression was analyzed in native oviductal fluid and oviduct epithelial cells (at different phases of the menstrual cycle: proliferative, periovulatory and secretory). In addition, the LF binding sites on spermatozoa (at different capacitation and acrosome reaction stages) and on ZP and the dose-dependent effect of LF on gamete interaction were investigated. All experiments were performed at least three times. Tubal tissues obtained from premenopausal patients (scheduled for hysterectomy, n = 23) were cultured in DMEM/Ham's F12 medium and conditioned media (CM) were collected. Motile spermatozoa were obtained by swim-up from normozoospermic semen samples from healthy donors (n = 4). An affinity chromatography with sperm membrane extracts was used to isolate proteins from CM. Isolated proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophresis and further identified by nano liquid chromatography tandem mass spectrometry peptide sequencing. The presence of LF in oviductal tissue was investigated by immunohistochemistry and immunofluorescence and was detected in native oviductal fluid and oviduct epithelial cells homogenates by western blot. LF binding sites on gametes were investigated by incubating gametes with the protein coupled to fluorescein isothiocyanate (FITC). The acrosome

Molecular mechanisms of Sonic hedgehog mutant effects in holoprosencephaly.

PubMed

Maity, Tapan; Fuse, Naoyuki; Beachy, Philip A

2005-11-22

Holoprosencephaly (HPE), a human developmental brain defect, usually is also associated with varying degrees of midline facial dysmorphism. Heterozygous mutations in the Sonic hedgehog (SHH) gene are the most common genetic lesions associated with HPE, and loss of Shh function in the mouse produces cyclopia and alobar forebrain development. The N-terminal domain (ShhNp) of Sonic hedgehog protein, generated by cholesterol-dependent autoprocessing and modification at the C terminus and by palmitate addition at the N terminus, is the active ligand in the Shh signal transduction pathway. Here, we analyze seven reported missense mutations (G31R, D88V, Q100H, N115K, W117G, W117R, and E188Q) that alter the N-terminal signaling domain of Shh protein, and show that two of these mutations (Q100H and E188Q), which are questionably linked to HPE, produce no detectable effects on function. The remaining five alterations affect normal processing, Ptc binding, and signaling to varying degrees. These effects include introduction of a recognition site for furin-like proteases by the G31R alteration, resulting in cleavage of 11 amino acid residues from the N terminus of ShhNp and consequent reduced signaling potency. Two other alterations, W117G and W117R, cause temperature-dependent misfolding and retention in the sterol-poor endoplasmic reticulum, thus disrupting cholesterol-dependent autoprocessing.

Functional convergence of Akt protein with VEGFR-1 in human endothelial progenitor cells exposed to sera from patient with type 2 diabetes mellitus.

PubMed

Hassanpour, Mehdi; Rezabakhsh, Aysa; Rahbarghazi, Reza; Nourazarian, Alireza; Nouri, Mohammad; Avci, Çığır Biray; Ghaderi, Shahrooz; Alidadyani, Neda; Bagca, Bakiye Goker; Bagheri, Hesam Saghaei

2017-11-01

Diabetes mellitus type 2 predisposes patients to various microvascular complications. In the current experiment, the potent role of diabetes mellitus was investigated on the content of VEGFR-1, -2, Tie-1 and -2, and Akt in human endothelial progenitor cells. The gene expression profile of mTOR and Hedgehog signaling pathways were measured by PCR array. The possible crosstalk between RTKs, mTOR and Hedgehog signaling was also studied by bioinformatic analysis. Endothelial progenitor cells were incubated with serum from normal and diabetic for 7days. Compared to non-treated cells, diabetic serum-induced cell apoptosis (~2-fold) and prohibited cell migration toward bFGF (p<0.001). ELISA analysis showed that diabetes exposed cells had increased abundance of Tie-1, -2 and VEGFR-2 and reduced amount of VEGFR-1 (p<0.0001) in diabetic cells. Western blotting showed a marked reduction in the protein level of Akt after cells exposure to serum from diabetic subjects (p<0.0001). PCR array revealed a significant stimulation of both mTOR and Hedgehog signaling pathways in diabetic cells (p<0.05). According to data from bioinformatic datasets, we showed VEGFR-1, -2 and Tie-2, but not Tie-1, are master regulators of angiogenesis. There is a crosstalk between RTKs and mTOR signaling by involving P62, GABARAPL1, and HTT genes. It seems that physical interaction and co-expression of Akt decreased the level of VEGFR-1 in diabetic cells. Regarding data from the present experiment, diabetic serum contributed to uncontrolled induction of both mTOR and Hedgehog signaling in endothelial progenitor cells. Diabetes mellitus induces mTOR pathway by involving receptor tyrosine kinases while Hedgehog stimulation is independent of these receptors. Copyright © 2017 Elsevier Inc. All rights reserved.

C2cd3 is required for cilia formation and Hedgehog signaling in mouse

PubMed Central

Hoover, Amber N.; Wynkoop, Aaron; Zeng, Huiqing; Jia, Jinping; Niswander, Lee A.; Liu, Aimin

2011-01-01

Cilia are essential for mammalian embryonic development as well as for the physiological activity of various adult organ systems. Despite the multiple crucial roles that cilia play, the mechanisms underlying ciliogenesis in mammals remain poorly understood. Taking a forward genetic approach, we have identified Hearty (Hty), a recessive lethal mouse mutant with multiple defects, including neural tube defects, abnormal dorsal-ventral patterning of the spinal cord, a defect in left-right axis determination and severe polydactyly (extra digits). By genetic mapping, sequence analysis of candidate genes and characterization of a second mutant allele, we identify Hty as C2cd3, a novel gene encoding a vertebrate-specific C2 domain-containing protein. Target gene expression and double-mutant analyses suggest that C2cd3 is an essential regulator of intracellular transduction of the Hedgehog signal. Furthering a link between Hedgehog signaling and cilia function, we find that cilia formation and proteolytic processing of Gli3 are disrupted in C2cd3 mutants. Finally, we observe C2cd3 protein at the basal body, consistent with its essential function in ciliogenesis. Interestingly, the human ortholog for this gene lies in proximity to the critical regions of Meckel-Gruber syndrome 2 (MKS2) and Joubert syndrome 2 (JBTS2), making it a potential candidate for these two human genetic disorders. PMID:19004860

Organization of the Indian hedgehog--parathyroid hormone-related protein system in the postnatal growth plate.

PubMed

Chau, Michael; Forcinito, Patricia; Andrade, Anenisia C; Hegde, Anita; Ahn, Sohyun; Lui, Julian C; Baron, Jeffrey; Nilsson, Ola

2011-08-01

In embryonic growth cartilage, Indian hedgehog (Ihh) and parathyroid hormone-related protein (PTHrP) participate in a negative feedback loop that regulates chondrocyte differentiation. Postnatally, this region undergoes major structural and functional changes. To explore the organization of the Ihh–PTHrP system in postnatal growth plate, we microdissected growth plates of 7-day-old rats into their constituent zones and assessed expression of genes participating in the h–PTHrP feedback loop. Ihh, Patched 1, Smoothened, Gli1, Gli2, Gli3, and Pthr1 were expressed in regions analogous to the expression domains in embryonic growth cartilage. However, PTHrP was expressed in resting zone cartilage, a site that differs from the embryonic source, the periarticular cells. We then used mice in which lacZ has replaced coding sequences of Gli1 and thus serves as a marker for active hedgehog signaling. At 1, 4, 8, and 12 weeks of age, lacZ expression was detected in a pattern analogous to that of embryonic cartilage. The findings support the hypothesis that the embryonic Ihh–PTHrP feedback loop is maintained in the postnatal growth plate except that the source of PTHrP has shifted to a more proximal location in the resting zone.

Interactions of the Human MCM-BP Protein with MCM Complex Components and Dbf4

PubMed Central

Nguyen, Tin; Jagannathan, Madhav; Shire, Kathy; Frappier, Lori

2012-01-01

MCM-BP was discovered as a protein that co-purified from human cells with MCM proteins 3 through 7; results which were recapitulated in frogs, yeast and plants. Evidence in all of these organisms supports an important role for MCM-BP in DNA replication, including contributions to MCM complex unloading. However the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood. Here we show that human MCM-BP is capable of interacting with individual MCM proteins 2 through 7 when co-expressed in insect cells and can greatly increase the recovery of some recombinant MCM proteins. Glycerol gradient sedimentation analysis indicated that MCM-BP interacts most strongly with MCM4 and MCM7. Similar gradient analyses of human cell lysates showed that only a small amount of MCM-BP overlapped with the migration of MCM complexes and that MCM complexes were disrupted by exogenous MCM-BP. In addition, large complexes containing MCM-BP and MCM proteins were detected at mid to late S phase, suggesting that the formation of specific MCM-BP complexes is cell cycle regulated. We also identified an interaction between MCM-BP and the Dbf4 regulatory component of the DDK kinase in both yeast 2-hybrid and insect cell co-expression assays, and this interaction was verified by co-immunoprecipitation of endogenous proteins from human cells. In vitro kinase assays showed that MCM-BP was not a substrate for DDK but could inhibit DDK phosphorylation of MCM4,6,7 within MCM4,6,7 or MCM2-7 complexes, with little effect on DDK phosphorylation of MCM2. Since DDK is known to activate DNA replication through phosphorylation of these MCM proteins, our results suggest that MCM-BP may affect DNA replication in part by regulating MCM phosphorylation by DDK. PMID:22540012

Interactions of the human MCM-BP protein with MCM complex components and Dbf4.

PubMed

Nguyen, Tin; Jagannathan, Madhav; Shire, Kathy; Frappier, Lori

2012-01-01

MCM-BP was discovered as a protein that co-purified from human cells with MCM proteins 3 through 7; results which were recapitulated in frogs, yeast and plants. Evidence in all of these organisms supports an important role for MCM-BP in DNA replication, including contributions to MCM complex unloading. However the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood. Here we show that human MCM-BP is capable of interacting with individual MCM proteins 2 through 7 when co-expressed in insect cells and can greatly increase the recovery of some recombinant MCM proteins. Glycerol gradient sedimentation analysis indicated that MCM-BP interacts most strongly with MCM4 and MCM7. Similar gradient analyses of human cell lysates showed that only a small amount of MCM-BP overlapped with the migration of MCM complexes and that MCM complexes were disrupted by exogenous MCM-BP. In addition, large complexes containing MCM-BP and MCM proteins were detected at mid to late S phase, suggesting that the formation of specific MCM-BP complexes is cell cycle regulated. We also identified an interaction between MCM-BP and the Dbf4 regulatory component of the DDK kinase in both yeast 2-hybrid and insect cell co-expression assays, and this interaction was verified by co-immunoprecipitation of endogenous proteins from human cells. In vitro kinase assays showed that MCM-BP was not a substrate for DDK but could inhibit DDK phosphorylation of MCM4,6,7 within MCM4,6,7 or MCM2-7 complexes, with little effect on DDK phosphorylation of MCM2. Since DDK is known to activate DNA replication through phosphorylation of these MCM proteins, our results suggest that MCM-BP may affect DNA replication in part by regulating MCM phosphorylation by DDK.

Recent coselection in human populations revealed by protein-protein interaction network.

PubMed

Qian, Wei; Zhou, Hang; Tang, Kun

2014-12-21

Genome-wide scans for signals of natural selection in human populations have identified a large number of candidate loci that underlie local adaptations. This is surprising given the relatively short evolutionary time since the divergence of the human population. One hypothesis that has not been formally examined is whether and how the recent human evolution may have been shaped by coselection in the context of complex molecular interactome. In this study, genome-wide signals of selection were scanned in East Asians, Europeans, and Africans using 1000 Genome data, and subsequently mapped onto the protein-protein interaction (PPI) network. We found that the candidate genes of recent positive selection localized significantly closer to each other on the PPI network than expected, revealing substantial clustering of selected genes. Furthermore, gene pairs of shorter PPI network distances showed higher similarities of their recent evolutionary paths than those further apart. Last, subnetworks enriched with recent coselection signals were identified, which are substantially overrepresented in biological pathways related to signal transduction, neurogenesis, and immune function. These results provide the first genome-wide evidence for association of recent selection signals with the PPI network, shedding light on the potential mechanisms of recent coselection in the human genome. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Interaction of perfluoroalkyl acids with human liver fatty acid-binding protein.

PubMed

Sheng, Nan; Li, Juan; Liu, Hui; Zhang, Aiqian; Dai, Jiayin

2016-01-01

Perfluoroalkyl acids (PFAAs) are highly persistent and bioaccumulative, resulting in their broad distribution in humans and the environment. The liver is an important target for PFAAs, but the mechanisms behind PFAAs interaction with hepatocyte proteins remain poorly understood. We characterized the binding of PFAAs to human liver fatty acid-binding protein (hL-FABP) and identified critical structural features in their interaction. The binding interaction of PFAAs with hL-FABP was determined by fluorescence displacement and isothermal titration calorimetry (ITC) assay. Molecular simulation was conducted to define interactions at the binding sites. ITC measurement revealed that PFOA/PFNA displayed a moderate affinity for hL-FABP at a 1:1 molar ratio, a weak binding affinity for PFHxS and no binding for PFHxA. Moreover, the interaction was mainly mediated by electrostatic attraction and hydrogen bonding. Substitution of Asn111 with Asp caused loss of binding affinity to PFAA, indicating its crucial role for the initial PFAA binding to the outer binding site. Substitution of Arg122 with Gly caused only one molecule of PFAA to bind to hL-FABP. Molecular simulation showed that substitution of Arg122 increased the volume of the outer binding pocket, making it impossible to form intensive hydrophobic stacking and hydrogen bonds with PFOA, and highlighting its crucial role in the binding process. The binding affinity of PFAAs increased significantly with their carbon number. Arg122 and Asn111 played a pivotal role in these interactions. Our findings may help understand the distribution pattern, bioaccumulation, elimination, and toxicity of PFAAs in humans.

Static flexural properties of hedgehog spines conditioned in coupled temperature and relative humidity environments.

PubMed

Kennedy, Emily B; Hsiung, Bor-Kai; Swift, Nathan B; Tan, Kwek-Tze

2017-11-01

Hedgehogs are agile climbers, scaling trees and plants to heights exceeding 10m while foraging insects. Hedgehog spines (a.k.a. quills) provide fall protection by absorbing shock and could offer insights for the design of lightweight, material-efficient, impact-resistant structures. There has been some study of flexural properties of hedgehog spines, but an understanding of how this keratinous biological material is affected by various temperature and relative humidity treatments, or how spine color (multicolored vs. white) affects mechanics, is lacking. To bridge this gap in the literature, we use three-point bending to analyze the effect of temperature, humidity, spine color, and their interactions on flexural strength and modulus of hedgehog spines. We also compare specific strength and stiffness of hedgehog spines to conventional engineered materials. We find hedgehog spine flexural properties can be finely tuned by modifying environmental conditioning parameters. White spines tend to be stronger and stiffer than multicolored spines. Finally, for most temperature and humidity conditioning parameters, hedgehog spines are ounce for ounce stronger than 201 stainless steel rods of the same diameter but as pliable as styrene rods with a slightly larger diameter. This unique combination of strength and elasticity makes hedgehog spines exemplary shock absorbers, and a suitable reference model for biomimicry. Copyright © 2017 Elsevier Ltd. All rights reserved.

Combination of Cyclopamine and Tamoxifen Promotes Survival and Migration of MCF-7 Breast Cancer Cells – Interaction of Hedgehog-Gli and Estrogen Receptor Signaling Pathways

PubMed Central

Uzarevic, Zvonimir; Ozretic, Petar; Musani, Vesna; Rafaj, Maja; Cindric, Mario; Levanat, Sonja

2014-01-01

Hedgehog-Gli (Hh-Gli) signaling pathway is one of the new molecular targets found upregulated in breast tumors. Estrogen receptor alpha (ERα) signaling has a key role in the development of hormone-dependent breast cancer. We aimed to investigate the effects of inhibiting both pathways simultaneously on breast cancer cell survival and the potential interactions between these two signaling pathways. ER-positive MCF-7 cells show decreased viability after treatment with cyclopamine, a Hh-Gli pathway inhibitor, as well as after tamoxifen (an ERα inhibitor) treatment. Simultaneous treatment with cyclopamine and tamoxifen on the other hand, causes short-term survival of cells, and increased migration. We found upregulated Hh-Gli signaling under these conditions and protein profiling revealed increased expression of proteins involved in cell proliferation and migration. Therefore, even though Hh-Gli signaling seems to be a good potential target for breast cancer therapy, caution must be advised, especially when combining therapies. In addition, we also show a potential direct interaction between the Shh protein and ERα in MCF-7 cells. Our data suggest that the Shh protein is able to activate ERα independently of the canonical Hh-Gli signaling pathway. Therefore, this may present an additional boost for ER-positive cells that express Shh, even in the absence of estrogen. PMID:25503972

Viral Organization of Human Proteins

PubMed Central

Wuchty, Stefan; Siwo, Geoffrey; Ferdig, Michael T.

2010-01-01

Although maps of intracellular interactions are increasingly well characterized, little is known about large-scale maps of host-pathogen protein interactions. The investigation of host-pathogen interactions can reveal features of pathogenesis and provide a foundation for the development of drugs and disease prevention strategies. A compilation of experimentally verified interactions between HIV-1 and human proteins and a set of HIV-dependency factors (HDF) allowed insights into the topology and intricate interplay between viral and host proteins on a large scale. We found that targeted and HDF proteins appear predominantly in rich-clubs, groups of human proteins that are strongly intertwined among each other. These assemblies of proteins may serve as an infection gateway, allowing the virus to take control of the human host by reaching protein pathways and diversified cellular functions in a pronounced and focused way. Particular transcription factors and protein kinases facilitate indirect interactions between HDFs and viral proteins. Discerning the entanglement of directly targeted and indirectly interacting proteins may uncover molecular and functional sites that can provide novel perspectives on the progression of HIV infection and highlight new avenues to fight this virus. PMID:20827298

Adding biological meaning to human protein-protein interactions identified by yeast two-hybrid screenings: A guide through bioinformatics tools.

PubMed

Felgueiras, Juliana; Silva, Joana Vieira; Fardilha, Margarida

2018-01-16

"A man is known by the company he keeps" is a popular expression that perfectly fits proteins. A common approach to characterize the function of a target protein is to identify its interacting partners and thus infer its roles based on the known functions of the interactors. Protein-protein interaction networks (PPINs) have been created for several organisms, including humans, primarily as results of high-throughput screenings, such as yeast two-hybrid (Y2H). Their unequivocal use to understand events underlying human pathophysiology is promising in identifying genes and proteins associated with diseases. Therefore, numerous opportunities have emerged for PPINs as tools for clinical management of diseases: network-based disease classification systems, discovery of biomarkers and identification of therapeutic targets. Despite the great advantages of PPINs, their use is still unrecognised by several researchers who generate high-throughput data to generally characterize interactions in a certain model or to select an interaction to study in detail. We strongly believe that both approaches are not exclusive and that we can use PPINs as a complementary methodology and rich-source of information to the initial study proposal. Here, we suggest a pipeline to deal with Y2H results using bioinformatics tools freely available for academics. Yeast two-hybrid is widely-used to identify protein-protein interactions. Conventionally, the positive clones that result from a yeast two-hybrid screening are sequenced to identify the interactors of the protein of interest (also known as bait protein), and few interactions, thought as potentially relevant for the model in study, are selected for further validation using biochemical methods (e.g. co-immunoprecipitation and co-localization). The huge amount of data that is potentially lost during this conservative approach motivated us to write this tutorial-like review, so that researchers feel encouraged to take advantage of

Clinical implications of hedgehog signaling pathway inhibitors

PubMed Central

Liu, Hailan; Gu, Dongsheng; Xie, Jingwu

2011-01-01

Hedgehog was first described in Drosophila melanogaster by the Nobel laureates Eric Wieschaus and Christiane Nüsslein-Volhard. The hedgehog (Hh) pathway is a major regulator of cell differentiation, proliferation, tissue polarity, stem cell maintenance, and Carcinogenesis. The first link of Hh signaling to cancer was established through studies of a rare familial disease, Gorlin syndrome, in 1996. Follow-up studies revealed activation of this pathway in basal cell carcinoma, medulloblastoma and, leukemia as well as in gastrointestinal, lung, ovarian, breast, and prostate cancer. Targeted inhibition of Hh signaling is now believed to be effective in the treatment and prevention of human cancer. The discovery and synthesis of specific inhibitors for this pathway are even more exciting. In this review, we summarize major advances in the understanding of Hh signaling pathway activation in human cancer, mouse models for studying Hh-mediated Carcinogenesis, the roles of Hh signaling in tumor development and metastasis, antagonists for Hh signaling and their clinical implications. PMID:21192841

Primary Cilia and Mammalian Hedgehog Signaling

PubMed Central

Bangs, Fiona; Anderson, Kathryn V.

2017-01-01

It has been a decade since it was discovered that primary cilia have an essential role in Hedgehog signaling in mammals. This discovery came from screens in the mouse that identified a set of genes that are required for both normal Hedgehog signaling and for the formation of primary cilia. Since then, dozens of mouse mutations have been identified that disrupt cilia in a variety of ways and have complex effects on Hedgehog signaling. Here we summarize the genetic and developmental studies used to deduce how Hedgehog signal transduction is linked to cilia and the complex effects that perturbation of cilia structure can have on Hh signaling. We conclude by describing the current status of our understanding of the cell-type specific regulation of ciliogenesis and how that determines the ability of cells to respond to Hedgehog ligands. PMID:27881449

A review of hedgehog signaling in cranial bone development

PubMed Central

Pan, Angel; Chang, Le; Nguyen, Alan; James, Aaron W.

2013-01-01

During craniofacial development, the Hedgehog (HH) signaling pathway is essential for mesodermal tissue patterning and differentiation. The HH family consists of three protein ligands: Sonic Hedgehog (SHH), Indian Hedgehog (IHH), and Desert Hedgehog (DHH), of which two are expressed in the craniofacial complex (IHH and SHH). Dysregulations in HH signaling are well documented to result in a wide range of craniofacial abnormalities, including holoprosencephaly (HPE), hypotelorism, and cleft lip/palate. Furthermore, mutations in HH effectors, co-receptors, and ciliary proteins result in skeletal and craniofacial deformities. Cranial suture morphogenesis is a delicate developmental process that requires control of cell commitment, proliferation and differentiation. This review focuses on both what is known and what remains unknown regarding HH signaling in cranial suture morphogenesis and intramembranous ossification. As demonstrated from murine studies, expression of both SHH and IHH is critical to the formation and fusion of the cranial sutures and calvarial ossification. SHH expression has been observed in the cranial suture mesenchyme and its precise function is not fully defined, although some postulate SHH to delay cranial suture fusion. IHH expression is mainly found on the osteogenic fronts of the calvarial bones, and functions to induce cell proliferation and differentiation. Unfortunately, neonatal lethality of IHH deficient mice precludes a detailed examination of their postnatal calvarial phenotype. In summary, a number of basic questions are yet to be answered regarding domains of expression, developmental role, and functional overlap of HH morphogens in the calvaria. Nevertheless, SHH and IHH ligands are integral to cranial suture development and regulation of calvarial ossification. When HH signaling goes awry, the resultant suite of morphologic abnormalities highlights the important roles of HH signaling in cranial development. PMID:23565096

From protein interaction profile to functional assignment: the human protein Ki-1/57 is associated with pre-mRNA splicing events.

PubMed

Bressan, Gustavo Costa; Kobarg, Jörg

2010-01-01

The mapping of protein-protein interactions of a determined organism is considered fundamental to assign protein function in the post-genomic era. As part of this effort, screenings for pairwise interactions by yeast two-hybrid system have been used popularly to reveal protein interaction networks in different biological systems. Through the identification of protein interaction partners we have successfully obtained interesting functional clues for Ki-1/57, a human protein with no previous functional annotation, in the context of RNA metabolism. We briefly discuss the way we approached protein-protein interaction data to conduct and interpret further molecular biological and cellular studies as well as structural analyses on this protein. Our data suggest that Ki-1/57 belongs to the family of intrinsically unstructured proteins and that the structural flexibility may be crucial for its capacity to interact with many different proteins. A large fraction of these proteins are involved in pre-mRNA splicing control. Finally, Ki-1/57 is localized to several subnuclear domains, all of which have been described to splicing and other RNA processing events.

HLPI-Ensemble: Prediction of human lncRNA-protein interactions based on ensemble strategy.

PubMed

Hu, Huan; Zhang, Li; Ai, Haixin; Zhang, Hui; Fan, Yetian; Zhao, Qi; Liu, Hongsheng

2018-03-27

LncRNA plays an important role in many biological and disease progression by binding to related proteins. However, the experimental methods for studying lncRNA-protein interactions are time-consuming and expensive. Although there are a few models designed to predict the interactions of ncRNA-protein, they all have some common drawbacks that limit their predictive performance. In this study, we present a model called HLPI-Ensemble designed specifically for human lncRNA-protein interactions. HLPI-Ensemble adopts the ensemble strategy based on three mainstream machine learning algorithms of Support Vector Machines (SVM), Random Forests (RF) and Extreme Gradient Boosting (XGB) to generate HLPI-SVM Ensemble, HLPI-RF Ensemble and HLPI-XGB Ensemble, respectively. The results of 10-fold cross-validation show that HLPI-SVM Ensemble, HLPI-RF Ensemble and HLPI-XGB Ensemble achieved AUCs of 0.95, 0.96 and 0.96, respectively, in the test dataset. Furthermore, we compared the performance of the HLPI-Ensemble models with the previous models through external validation dataset. The results show that the false positives (FPs) of HLPI-Ensemble models are much lower than that of the previous models, and other evaluation indicators of HLPI-Ensemble models are also higher than those of the previous models. It is further showed that HLPI-Ensemble models are superior in predicting human lncRNA-protein interaction compared with previous models. The HLPI-Ensemble is publicly available at: http://ccsipb.lnu.edu.cn/hlpiensemble/ .

Cardiomyopathy in captive African hedgehogs (Atelerix albiventris).

PubMed

Raymond, J T; Garner, M M

2000-09-01

From 1994 to 1999, 16 captive African hedgehogs (Atelerix albiventris), from among 42 necropsy cases, were diagnosed with cardiomyopathy. The incidence of cardiomyopathy in this study population was 38%. Fourteen of 16 hedgehogs with cardiomyopathy were males and all hedgehogs were adult (>1 year old). Nine hedgehogs exhibited 1 or more of the following clinical signs before death: heart murmur, lethargy, icterus, moist rales, anorexia, dyspnea, dehydration, and weight loss. The remaining 7 hedgehogs died without premonitory clinical signs. Gross findings were cardiomegaly (6 cases), hepatomegaly (5 cases), pulmonary edema (5 cases), pulmonary congestion (4 cases), hydrothorax (3 cases), pulmonary infarct (1 case), renal infarcts (1 case), ascites (1 case), and 5 cases showed no changes. Histologic lesions were found mainly within the left ventricular myocardium and consisted primarily of myodegeneration, myonecrosis, atrophy, hypertrophy, and disarray of myofibers. All hedgehogs with cardiomyopathy had myocardial fibrosis, myocardial edema, or both. Other common histopathologic findings were acute and chronic passive congestion of the lungs, acute passive congestion of the liver, renal tubular necrosis, vascular thrombosis, splenic extramedullary hematopoiesis, and hepatic lipidosis. This is the first report of cardiomyopathy in African hedgehogs.

Hedgehog pathway as a potential treatment target in human cholangiocarcinoma.

PubMed

Riedlinger, Dorothee; Bahra, Marcus; Boas-Knoop, Sabine; Lippert, Steffen; Bradtmöller, Maren; Guse, Katrin; Seehofer, Daniel; Bova, Roberta; Sauer, Igor M; Neuhaus, Peter; Koch, Arend; Kamphues, Carsten

2014-08-01

Innovative treatment concepts targeting essential signaling pathways may offer new chances for patients suffering from cholangiocarcinoma (CCC). For that, we performed a systematic molecular genetic analysis concerning the Hedgehog activity in human CCC samples and analyzed the effect of Hh inhibition on CCC cells in vitro and in vivo. Activation of the Hh pathway was analyzed in 50 human CCC samples using quantitative polymerase chain reaction (qPCR). The efficacy of Hh inhibition using cyclopamine and BMS-833923 was evaluated in vitro. In addition, the effect of BMS-833923, alone or in combination with gemcitabine, was analyzed in vivo in a murine subcutaneous xenograft model. Expression analysis revealed a significant activation of the Hh-signaling pathway in nearly 50% of CCCs. Hh inhibition resulted in a significant decrease in cell proliferation of CCC cells. Moreover, a distinct inhibition of tumor growth could be seen as a result of a combined therapy with BMS-833923 and gemcitabine in CCC xenografts. The results of our study suggest that the Hh pathway plays a relevant role at least in a subset of human CCC. Inhibition of this pathway may represent a possible treatment option for CCC patients in which the Hh pathway is activated. © 2014 Japanese Society of Hepato-Biliary-Pancreatic Surgery.

Position Matters: Network Centrality Considerably Impacts Rates of Protein Evolution in the Human Protein–Protein Interaction Network

PubMed Central

Feyertag, Felix; Chakraborty, Sandip

2017-01-01

Abstract The proteins of any organism evolve at disparate rates. A long list of factors affecting rates of protein evolution have been identified. However, the relative importance of each factor in determining rates of protein evolution remains unresolved. The prevailing view is that evolutionary rates are dominantly determined by gene expression, and that other factors such as network centrality have only a marginal effect, if any. However, this view is largely based on analyses in yeasts, and accurately measuring the importance of the determinants of rates of protein evolution is complicated by the fact that the different factors are often correlated with each other, and by the relatively poor quality of available functional genomics data sets. Here, we use correlation, partial correlation and principal component regression analyses to measure the contributions of several factors to the variability of the rates of evolution of human proteins. For this purpose, we analyzed the entire human protein–protein interaction data set and the human signal transduction network—a network data set of exceptionally high quality, obtained by manual curation, which is expected to be virtually free from false positives. In contrast with the prevailing view, we observe that network centrality (measured as the number of physical and nonphysical interactions, betweenness, and closeness) has a considerable impact on rates of protein evolution. Surprisingly, the impact of centrality on rates of protein evolution seems to be comparable, or even superior according to some analyses, to that of gene expression. Our observations seem to be independent of potentially confounding factors and from the limitations (biases and errors) of interactomic data sets. PMID:28854629

Detection of a pneumonia virus of mice (PVM) in an African hedgehog (Atelerix arbiventris) with suspected wobbly hedgehog syndrome (WHS).

PubMed

Madarame, Hiroo; Ogihara, Kikumi; Kimura, Moe; Nagai, Makoto; Omatsu, Tsutomu; Ochiai, Hideharu; Mizutani, Tetsyuya

2014-09-17

A pneumonia virus of mice (PVM) from an African hedgehog (Atelerix arbiventris) with suspected wobbly hedgehog syndrome (WHS) was detected and genetically characterized. The affected hedgehog had a nonsuppurative encephalitis with vacuolization of the white matter, and the brain samples yielded RNA reads highly homogeneous to PVM strain 15 (96.5% of full genomic sequence homology by analysis of next generation sequencing). PVM antigen was also detected in the brain and the lungs immunohistochemically. A PVM was strongly suggested as a causative agent of encephalitis of a hedgehog with suspected WHS. This is a first report of PVM infection in hedgehogs. Copyright © 2014 Elsevier B.V. All rights reserved.

Positive and Negative Regulation of Muscle Cell Identity by Members of the hedgehog and TGF-β Gene Families

PubMed Central

Du, Shao Jun; Devoto, Stephen H.; Westerfield, Monte; Moon, Randall T.

1997-01-01

We have examined whether the development of embryonic muscle fiber type is regulated by competing influences between Hedgehog and TGF-β signals, as previously shown for development of neuronal cell identity in the neural tube. We found that ectopic expression of Hedgehogs or inhibition of protein kinase A in zebrafish embryos induces slow muscle precursors throughout the somite but muscle pioneer cells only in the middle of the somite. Ectopic expression in the notochord of Dorsalin-1, a member of the TGF-β superfamily, inhibits the formation of muscle pioneer cells, demonstrating that TGF-β signals can antagonize the induction of muscle pioneer cells by Hedgehog. We propose that a Hedgehog signal first induces the formation of slow muscle precursor cells, and subsequent Hedgehog and TGF-β signals exert competing positive and negative influences on the development of muscle pioneer cells. PMID:9314535

Intestinal lymphosarcoma in captive African hedgehogs.

PubMed

Raymond, J T; Clarke, K A; Schafer, K A

1998-10-01

Two captive adult female African hedgehogs (Atelerix albiventris) had inappetance and bloody diarrhea for several days prior to death. Both hedgehogs had ulceration of the small intestine and hepatic lipidosis. Histopathology revealed small intestinal lymphosarcoma with metastasis to the liver. Extracellular particles that had characteristics of retroviruses were observed associated with the surface of some neoplastic lymphoid cells by transmission electron microscopy. These are the first reported cases of intestinal lymphosarcoma in African hedgehogs.

[Oligodendroglioma with neuronal differentiation in an 8-month-old African hedgehog (Atelerix albiventris)].

PubMed

Völker, Iris; Schwarze, Iris; Brezina, Tina E; Köstlinger, Saskia; Hewicker-Trautwein, Marion

2016-10-12

An 8-month-old, male African hedgehog clinically displayed a wobbly walk, anuria, inappetence and apathy, whereupon the suspected diagnosis wobbly hedgehog syndrome was made. After exacerbation, the hedgehog was euthanized. Histologically, a tumour mainly consisting of medium-sized, oval tumour cells and a smaller number of spindeloid cells was found in the cerebrum. The tumour contained neuropil islets and extracellular myxoid material. Immunohistochemically, the tumour cells expressed oligodendroglial (neurite outgrowth inhibitor, Nogo-A; oligodendrocyte transcription factor, Olig-2) and neuronal (neuron-specific enolase, NSE; microtubule-associated protein-2a, MAP-2a; synaptophysin) cell markers. Based on these findings, an oligodendroglioma with neuronal differentiation was diagnosed. Such a brain tumour has to date not been reported for African hedgehogs. At necropsy, a severely filled and dilated urinary bladder was observed, which was presumably caused by a central blockade of the micturition centre in the brain. In the case of neurological symptoms in young hedgehogs, a primary brain tumour should, as in adults, be considered as a differential diagnosis. As further differentials, inflammatory-infectious (rabies, herpes, baylisascariosis), degenerative (cardiomyopathy, intervertebral-disc disease), traumatic, alimentary (vitamin-B deficiency) and metabolic-toxic (heat-cold-torpor, hepatic encephalopathy) triggers have to be considered.

A yeast 2-hybrid analysis of human GTP cyclohydrolase I protein interactions

PubMed Central

Swick, Lance; Kapatos, Gregory

2008-01-01

The yeast 2-hybrid system was used to identify protein domains involved in the oligomerization of human guanosine 5′-triphosphate (GTP) Cyclohydrolase I (GCH1) and the interaction of GCH1 with its regulatory partner, GCH1 feedback regulatory protein (GFRP). When interpreted within the structural framework derived from crystallography, our results indicate that the GCH1 N-terminal α-helices are not the only domains involved in the formation of dimers from monomers and also suggest an important role for the C-terminal α-helix in the assembly of dimers to form decamers. Moreover, a previously unknown role of the extended N-terminal α–helix in the interaction of GCH1 and GFRP was revealed. To discover novel GCH1 protein binding partners, we used the yeast 2-hybrid system to screen a human brain library with GCH1 N-terminal amino acids 1–96 as prey. This protruding extension of GCH1 contains two canonical Type-I Src homology-3 (SH3) ligand domains located within amino acids 1–42. Our screen yielded seven unique clones that were subsequently shown to require amino acids 1–42 for binding to GCH1. The interaction of one of these clones, Activator of Heat Shock 90 kDa Protein (Aha1), with GCH1 was validated by glutathione-s-transferase (GST) pull-down assay. Although the physiological relevance of the Aha1–GCH1 interaction requires further study, Aha1 may recruit GCH1 into the endothelial nitric oxide synthase/heat shock protein (eNOS/Hsp90) complex to support changes in endothelial nitric oxide production through the local synthesis of BH4. PMID:16696853

Trichophyton erinacei in pet hedgehogs in Spain: Occurrence and revision of its taxonomic status.

PubMed

Abarca, M L; Castellá, G; Martorell, J; Cabañes, F J

2017-02-01

Hedgehogs have increased in popularity as pets in Spain but there are no data of infection rates of this exotic animal with dermatophytes in our country. During the period of 2008-2011 a total of 20 pet hedgehogs (19 African pygmy hedgehogs and 1 Egyptian long-eared hedgehog) suspected of having dermatophytoses were studied. This is the first survey of the occurrence of T. erinacei in household hedgehogs in Spain. The T. erinacei infection rate was 50% (9 out of 19 African pygmy hedgehogs, and the one Egyptian long-eared hedgehog surveyed). Morphological identification of the isolates was confirmed by molecular analysis. All the strains had the same ITS sequence and showed 100% sequence similarity to T. erinacei type strain CBS 511.73 (AB 105793). The Spanish isolates were confirmed as T. erinacei urease positive. On the basis of ITS sequences, T. erinacei is a species close to but separate from the taxa included in the A. benhamiae complex. Review of the current literature on DNA-based methods for identification of species included in this complex has highlighted the urgent need to reach a consensus in species circumscription and classification system accepted by all mycologists. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Prediction of virus-host protein-protein interactions mediated by short linear motifs.

PubMed

Becerra, Andrés; Bucheli, Victor A; Moreno, Pedro A

2017-03-09

Short linear motifs in host organisms proteins can be mimicked by viruses to create protein-protein interactions that disable or control metabolic pathways. Given that viral linear motif instances of host motif regular expressions can be found by chance, it is necessary to develop filtering methods of functional linear motifs. We conduct a systematic comparison of linear motifs filtering methods to develop a computational approach for predicting motif-mediated protein-protein interactions between human and the human immunodeficiency virus 1 (HIV-1). We implemented three filtering methods to obtain linear motif sets: 1) conserved in viral proteins (C), 2) located in disordered regions (D) and 3) rare or scarce in a set of randomized viral sequences (R). The sets C,D,R are united and intersected. The resulting sets are compared by the number of protein-protein interactions correctly inferred with them - with experimental validation. The comparison is done with HIV-1 sequences and interactions from the National Institute of Allergy and Infectious Diseases (NIAID). The number of correctly inferred interactions allows to rank the interactions by the sets used to deduce them: D∪R and C. The ordering of the sets is descending on the probability of capturing functional interactions. With respect to HIV-1, the sets C∪R, D∪R, C∪D∪R infer all known interactions between HIV1 and human proteins mediated by linear motifs. We found that the majority of conserved linear motifs in the virus are located in disordered regions. We have developed a method for predicting protein-protein interactions mediated by linear motifs between HIV-1 and human proteins. The method only use protein sequences as inputs. We can extend the software developed to any other eukaryotic virus and host in order to find and rank candidate interactions. In future works we will use it to explore possible viral attack mechanisms based on linear motif mimicry.

Cholesterol activates the G-protein coupled receptor Smoothened to promote Hedgehog signaling

PubMed Central

Luchetti, Giovanni; Sircar, Ria; Kong, Jennifer H; Nachtergaele, Sigrid; Sagner, Andreas; Byrne, Eamon FX; Covey, Douglas F; Siebold, Christian; Rohatgi, Rajat

2016-01-01

Cholesterol is necessary for the function of many G-protein coupled receptors (GPCRs). We find that cholesterol is not just necessary but also sufficient to activate signaling by the Hedgehog (Hh) pathway, a prominent cell-cell communication system in development. Cholesterol influences Hh signaling by directly activating Smoothened (SMO), an orphan GPCR that transmits the Hh signal across the membrane in all animals. Unlike many GPCRs, which are regulated by cholesterol through their heptahelical transmembrane domains, SMO is activated by cholesterol through its extracellular cysteine-rich domain (CRD). Residues shown to mediate cholesterol binding to the CRD in a recent structural analysis also dictate SMO activation, both in response to cholesterol and to native Hh ligands. Our results show that cholesterol can initiate signaling from the cell surface by engaging the extracellular domain of a GPCR and suggest that SMO activity may be regulated by local changes in cholesterol abundance or accessibility. DOI: http://dx.doi.org/10.7554/eLife.20304.001 PMID:27705744

Ticks and Fleas Infestation on East Hedgehogs (Erinaceus concolor) in Van Province, Eastern Region of Turkey

PubMed Central

Goz, Yaşar; Yilmaz, Ali Bilgin; Aydin, Abdulalim; Dicle, Yalçın

2016-01-01

Background: Ixodid ticks (Acari: İxodidae) and fleas (Siphonaptera) are the major vectors of pathogens threatening animals and human healths. The aim of our study was to detect the infestation rates of East Hedgehogs (Erinaceus concolor) with ticks and fleas in Van Province, eastern region of Turkey. Methods: We examined fleas and ticks infestation patterns in 21 hedgehogs, collected from three suburbs with the greater of number gardens. In order to estimate flea and tick infestation of hedgehogs, we immobilized the ectoparasites by treatment the body with a insecticide trichlorphon (Neguvon®-Bayer). Results: On the hedgehogs, 60 ixodid ticks and 125 fleas were detected. All of the ixodid ticks were Rhipicephalus turanicus and all of the fleas were Archaeopsylla erinacei. Infestation rate for ticks and fleas was detected 66.66 % and 100 %, respectively. Conclusion: We detected ticks (R. turanicus) and fleas (A. erinacei) in hedgehogs at fairly high rates. Since many ticks and fleas species may harbor on hedgehogs and transmit some tick-borne and flea-borne patogens, this results are the important in terms of veterinary and public health. PMID:27047971

Noxa/Bcl-2 Protein Interactions Contribute to Bortezomib Resistance in Human Lymphoid Cells*

PubMed Central

Smith, Alyson J.; Dai, Haiming; Correia, Cristina; Takahashi, Rie; Lee, Sun-Hee; Schmitz, Ingo; Kaufmann, Scott H.

2011-01-01

Previous studies have suggested that the BH3 domain of the proapoptotic Bcl-2 family member Noxa only interacts with the anti-apoptotic proteins Mcl-1 and A1 but not Bcl-2. In view of the similarity of the BH3 binding domains of these anti-apoptotic proteins as well as recent evidence that studies of isolated BH3 domains can potentially underestimate the binding between full-length Bcl-2 family members, we examined the interaction of full-length human Noxa with anti-apoptotic human Bcl-2 family members. Surface plasmon resonance using bacterially expressed proteins demonstrated that Noxa binds with mean dissociation constants (KD) of 3.4 nm for Mcl-1, 70 nm for Bcl-xL, and 250 nm for wild type human Bcl-2, demonstrating selectivity but not absolute specificity of Noxa for Mcl-1. Further analysis showed that the Noxa/Bcl-2 interaction reflected binding between the Noxa BH3 domain and the Bcl-2 BH3 binding groove. Analysis of proteins expressed in vivo demonstrated that Noxa and Bcl-2 can be pulled down together from a variety of cells. Moreover, when compared with wild type Bcl-2, certain lymphoma-derived Bcl-2 mutants bound Noxa up to 20-fold more tightly in vitro, pulled down more Noxa from cells, and protected cells against killing by transfected Noxa to a greater extent. When killing by bortezomib (an agent whose cytotoxicity in Jurkat T-cell leukemia cells is dependent on Noxa) was examined, apoptosis was enhanced by the Bcl-2/Bcl-xL antagonist ABT-737 or by Bcl-2 down-regulation and diminished by Bcl-2 overexpression. Collectively, these observations not only establish the ability of Noxa and Bcl-2 to interact but also identify Bcl-2 overexpression as a potential mechanism of bortezomib resistance. PMID:21454712

Emilin3 is required for notochord sheath integrity and interacts with Scube2 to regulate notochord-derived Hedgehog signals.

PubMed

Corallo, Diana; Schiavinato, Alvise; Trapani, Valeria; Moro, Enrico; Argenton, Francesco; Bonaldo, Paolo

2013-11-01

The notochord is a transient and essential structure that provides both mechanical and signaling cues to the developing vertebrate embryo. In teleosts, the notochord is composed of a core of large vacuolated cells and an outer layer of cells that secrete the notochord sheath. In this work, we have identified the extracellular matrix glycoprotein Emilin3 as a novel essential component of the zebrafish notochord sheath. The development of the notochord sheath is impaired in Emilin3 knockdown embryos. The patterning activity of the notochord is also affected by Emilin3, as revealed by the increase of Hedgehog (Hh) signaling in Emilin3-depleted embryos and the decreased Hh signaling in embryos overexpressing Emilin3 in the notochord. In vitro and in vivo experiments indicate that Emilin3 modulates the availability of Hh ligands by interacting with the permissive factor Scube2 in the notochord sheath. Overall, this study reveals a new role for an EMILIN protein and reinforces the concept that structure and function of the notochord are strictly linked.

Sarcoptes scabiei on hedgehogs in New Zealand.

PubMed

Kriechbaum, Caroline; Pomroy, William; Gedye, Kristene

2018-03-01

European hedgehogs (Erinaceus europaeus) were introduced into New Zealand from Britain during the period from 1869 to the early 1900s. The only mite found on New Zealand hedgehogs in early studies was Caparinia tripilis, with Sarcoptes scabiei first being reported in 1996. The aim of this study was to investigate the prevalence of Sarcoptes infestation on hedgehogs in New Zealand, the number of mites found and the degree of mange observed. Dead hedgehogs were collected from veterinary clinics, rescue centres, members of the public and from road-kill. Twenty-one (55.3%) of the animals examined had visible skin lesions. Both Caparinia and Sarcoptes mites were identified on microscopic examination with Sarcoptes the most common, being found on over 70% of animals examined (n = 38). The numbers of mites recovered after brushing the head and body ranged from 1 to 5659 (median = 341 mites) with only six animals (22.2%) having fewer than 10 Sarcoptes mites found. Caparinia mites were seen on fewer animals and generally in very low numbers. These findings indicate a change in the mite populations on hedgehogs in New Zealand and that infected animals develop the debilitating hyperkeratotic form of sarcoptic mange without an accompanying hypersensitivity response limiting numbers of mites. Analysis of the cox 1 gene of Sarcoptes from two hedgehogs showed close alignment to sequences derived from a pig with one and from a dog with the second. More work needs to be undertaken to identify the source(s) of the Sarcoptes found on hedgehogs in New Zealand and whether other mammalian hosts may be infected from contact with hedgehogs.

Human HOX Proteins Use Diverse and Context-Dependent Motifs to Interact with TALE Class Cofactors.

PubMed

Dard, Amélie; Reboulet, Jonathan; Jia, Yunlong; Bleicher, Françoise; Duffraisse, Marilyne; Vanaker, Jean-Marc; Forcet, Christelle; Merabet, Samir

2018-03-13

HOX proteins achieve numerous functions by interacting with the TALE class PBX and MEIS cofactors. In contrast to this established partnership in development and disease, how HOX proteins could interact with PBX and MEIS remains unclear. Here, we present a systematic analysis of HOX/PBX/MEIS interaction properties, scanning all paralog groups with human and mouse HOX proteins in vitro and in live cells. We demonstrate that a previously characterized HOX protein motif known to be critical for HOX-PBX interactions becomes dispensable in the presence of MEIS in all except the two most anterior paralog groups. We further identify paralog-specific TALE-binding sites that are used in a highly context-dependent manner. One of these binding sites is involved in the proliferative activity of HOXA7 in breast cancer cells. Together these findings reveal an extraordinary level of interaction flexibility between HOX proteins and their major class of developmental cofactors. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Ectoparasites infestation of free-ranging hedgehog (Etelerix algirus) in north western Libya

PubMed Central

Hosni, M.M.; Maghrbi, A.A. El

2014-01-01

The aim of this study was to assess the prevalence of ectoparasites in hedgehogs (Etelerix algirus) in north western region of Libya. Seventy hedgehogs were sampled, and 39 (55.7%) were infested with external parasites. A total of 44 ticks, 491 fleas were collected from the infested hedgehogs and four species of ectoparasites were identified, one mite (Sarcoptes scabiei), one tick (Rhipicephalus appendiculatus) and two fleas (Xenopsylla cheopis and Ctenocephalides canis). For ectoparasites, 10/39 (25.6%) were infested by S. scabiei, 8/39 (20.5%) by Rh. appendiculatus and 11/39 (28.2%) by fleas. The prevalence of mixed infestation with S. scabiei and C. canis was 3(7.7%), Rh. appendiculatus and C. canis was 2 (5.1%) and infestation by two species of fleas was 5 (12.8%). The overall mixed infestation was 10 (25.6%). We concluded that the hedgehogs may play an important role in spreading external parasites and transmission of diseases from one region to another and from wildlife animals to domestic animals and human. PMID:26623333

Mammary gland tumors in captive African hedgehogs.

PubMed

Raymond, J T; Gerner, M

2000-04-01

From December 1995 to July 1999, eight mammary gland tumors were diagnosed in eight adult captive female African hedgehogs (Atelerix albiventris). The tumors presented as single or multiple subcutaneous masses along the cranial or caudal abdomen that varied in size for each hedgehog. Histologically, seven of eight (88%) mammary gland tumors were malignant. Tumors were classified as solid (4 cases), tubular (2 cases), and papillary (2 cases). Seven tumors had infiltrated into the surrounding stroma and three tumors had histologic evidence of neoplastic vascular invasion. Three hedgehogs had concurrent neoplasms. These are believed to be the first reported cases of mammary gland tumors in African hedgehogs.

Protein-protein interaction studies reveal the Plasmodium falciparum merozoite surface protein-1 region involved in a complex formation that binds to human erythrocytes.

PubMed

Paul, Gourab; Deshmukh, Arunaditya; Kumar Chourasia, Bishwanath; Kalamuddin, Md; Panda, Ashutosh; Kumar Singh, Susheel; Gupta, Puneet K; Mohmmed, Asif; Chauhan, Virender S; Theisen, Michael; Malhotra, Pawan

2018-03-29

Plasmodium falciparum merozoite surface protein (PfMSP) 1 has been studied extensively as a vaccine candidate antigen. PfMSP-1 undergoes proteolytic processing into four major products, such as p83, p30, p38, and p42, that are associated in the form of non-covalent complex(s) with other MSPs. To delineate MSP1 regions involved in the interaction with other MSPs, here we expressed recombinant proteins (PfMSP-1 65 ) encompassing part of p38 and p42 regions and PfMSP-1 19 PfMSP-1 65 interacted strongly with PfMSP-3, PfMSP-6, PfMSP-7, and PfMSP-9, whereas PfMSP-1 19 did not interact with any of these proteins. Since MSP-1 complex binds human erythrocytes, we examined the ability of these proteins to bind human erythrocyte. Among the proteins of MSP-1 complex, PfMSP-6 and PfMSP-9 bound to human erythrocytes. Serological studies showed that PfMSP-1 65 was frequently recognized by sera from malaria endemic regions, whereas this was not the case for PfMSP-1 19 In contrast, antibodies against PfMSP-1 19 showed much higher inhibition of merozoite invasion compared with antibodies against the larger PfMSP-1 65 fragment. Importantly, anti-PfMSP-1 19 antibodies recognized both recombinant proteins, PfMSP-1 19 and PfMSP-1 65 ; however, anti-PfMSP-1 65 antibody failed to recognize the PfMSP-1 19 protein. Taken together, these results demonstrate that PfMSP-1 sequences upstream of the 19 kDa C-terminal region are involved in molecular interactions with other MSPs, and these sequences may probably serve as a smoke screen to evade antibody response to the membrane-bound C-terminal 19 kDa region. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Disseminated histoplasmosis in an African pygmy hedgehog.

PubMed

Snider, Timothy A; Joyner, Priscilla H; Clinkenbeard, Kenneth D

2008-01-01

A 2-year-old captive-bred sexually intact female African pygmy hedgehog (Atelerix albiventris) was evaluated because of vague signs of illness including inappetence, weakness, lethargy, and weight loss over a 20-day period. Abnormalities detected via initial clinicopathologic analyses included anemia, thrombocytopenia, leukopenia, hypoproteinemia, and hypoglycemia. Results of a fecal flotation test were negative. Three weeks after the initial evaluation, splenomegaly was detected via palpation and ultrasonography. The hedgehog was treated with broad-spectrum antibacterial agents, resulting in an initially favorable response. Fenbendazole was also administered against possible occult parasitic infestation. After 3 weeks of illness, the hedgehog's condition had worsened and supportive care and administration of additional antibacterial agents were instituted. The hedgehog died, and pathologic examinations revealed severe splenomegaly; granulomatous infiltrates were evident in multiple organs, and Histoplasma capsulatum yeasts were detected intralesionally. Histoplasmosis can develop in a wide range of mammalian species. African pygmy hedgehogs are becoming increasingly popular as exotic pets, and vague signs of illness and splenomegaly are often attributed to hemolymphatic malignancies, which are somewhat common in this species. Practitioners should be aware that similar clinical signs may be associated with histoplasmosis in these animals. Although the hedgehog of this report was confined indoors, it originated from an area where histoplasmosis was endemic; this indicates that the disease should be included as a differential diagnosis for hedgehogs that develop vague signs of illness and are known to originate from such geographic regions.

Gedunin inhibits pancreatic cancer by altering sonic hedgehog signaling pathway

PubMed Central

Subramani, Ramadevi; Gonzalez, Elizabeth; Nandy, Sushmita Bose; Arumugam, Arunkumar; Camacho, Fernando; Medel, Joshua; Alabi, Damilola; Lakshmanaswamy, Rajkumar

2017-01-01

INTRODUCTION The lack of efficient treatment options for pancreatic cancer highlights the critical need for the development of novel and effective chemotherapeutic agents. The medicinal properties found in plants have been used to treat many different illnesses including cancers. This study focuses on the anticancer effects of gedunin, a natural compound isolated from Azadirachta indica. METHODS Anti–proliferative effect of gedunin on pancreatic cancer cells was assessed using MTS assay. We used matrigel invasion assay, scratch assay, and soft agar colony formation assay to measure the anti–metastatic potential of gedunin. Immunoblotting was performed to analyze the effect of gedunin on the expression of key proteins involved in pancreatic cancer growth and metastasis. Gedunin induced apoptosis was measured using flow cytometric analysis. To further validate, xenograft studies with HPAC cells were performed. RESULTS Gedunin treatment is highly effective in inducing death of pancreatic cancer cells via intrinsic and extrinsic mediated apoptosis. Our data further indicates that gedunin inhibited metastasis of pancreatic cancer cells by decreasing their EMT, invasive, migratory and colony formation capabilities. Gedunin treatment also inhibited sonic hedgehog signaling pathways. Further, experiments with recombinant sonic hedgehog protein and Gli inhibitor (Gant-61) demonstrated that gedunin induces its anti–metastatic effect through inhibition of sonic hedgehog signaling. The anti–cancer effect of gedunin was further validated using xenograft mouse model. CONCLUSION Overall, our data suggests that gedunin could serve as a potent anticancer agent against pancreatic cancers. PMID:26988754

Interaction Network of Proteins Associated with Human Cytomegalovirus IE2-p86 Protein during Infection: A Proteomic Analysis

PubMed Central

Du, Guixin; Stinski, Mark F.

2013-01-01

Human cytomegalovirus protein IE2-p86 exerts its functions through interaction with other viral and cellular proteins. To further delineate its protein interaction network, we generated a recombinant virus expressing SG-tagged IE2-p86 and used tandem affinity purification coupled with mass spectrometry. A total of 9 viral proteins and 75 cellular proteins were found to associate with IE2-p86 protein during the first 48 hours of infection. The protein profile at 8, 24, and 48 h post infection revealed that UL84 tightly associated with IE2-p86, and more viral and cellular proteins came into association with IE2-p86 with the progression of virus infection. A computational analysis of the protein-protein interaction network indicated that all of the 9 viral proteins and most of the cellular proteins identified in the study are interconnected to varying degrees. Of the cellular proteins that were confirmed to associate with IE2-p86 by immunoprecipitation, C1QBP was further shown to be upregulated by HCMV infection and colocalized with IE2-p86, UL84 and UL44 in the virus replication compartment of the nucleus. The IE2-p86 interactome network demonstrated the temporal development of stable and abundant protein complexes that associate with IE2-p86 and provided a framework to benefit future studies of various protein complexes during HCMV infection. PMID:24358118

Prevalence and characterization of Salmonella enterica from the feces of cattle, poultry, swine and hedgehogs in Burkina Faso and their comparison to human Salmonella isolates

PubMed Central

2013-01-01

Background Production and wild animals are major sources of human salmonellosis and animals raised for food also play an important role in transmission of antimicrobial resistant Salmonella strains to humans. Furthermore, in sub-Saharan Africa non-typhoidal Salmonella serotypes are common bloodstream isolates in febrile patients. Yet, little is known about the environmental reservoirs and predominant modes of transmission of these pathogens. The purpose of this study was to discover potential sources and distribution vehicles of Salmonella by isolating strains from apparently healthy slaughtered food animals and wild hedgehogs and by determining the genetic relatedness between the strains and human isolates. For this purpose, 729 feces samples from apparently healthy slaughtered cattle (n = 304), poultry (n = 350), swine (n = 50) and hedgehogs (n = 25) were examined for the presence of Salmonella enterica in Burkina Faso. The isolates were characterized by serotyping, antimicrobial-susceptibility testing, phage typing, and pulsed-field gel electrophoresis (PFGE) with XbaI and BlnI restriction enzymes. Results Of the 729 feces samples, 383 (53%) contained Salmonella, representing a total of 81 different serotypes. Salmonella was present in 52% of the cattle, 55% of the poultry, 16% of the swine and 96% of the hedgehog feces samples. Antimicrobial resistance was detected in 14% of the isolates. S. Typhimurium isolates from poultry and humans (obtained from a previous study) were multiresistant to the same antimicrobials (ampicillin, chloramphenicol, streptomycin, sulfonamides and trimethoprim), had the same phage type DT 56 and were closely related in PFGE. S. Muenster isolates from hedgehogs had similar PFGE patterns as the domestic animals. Conclusions Based on our results it seems that production and wild animals can share the same Salmonella serotypes and potentially transmit some of them to humans. As the humans and animals often live in close

Could an endoneurial endothelial crosstalk between Wnt/β-catenin and Sonic Hedgehog pathways underlie the early disruption of the infra-orbital blood-nerve barrier following chronic constriction injury?

PubMed

Moreau, Nathan; Mauborgne, Annie; Couraud, Pierre-Olivier; Romero, Ignacio A; Weksler, Babette B; Villanueva, Luis; Pohl, Michel; Boucher, Yves

2017-01-01

Blood–nerve barrier disruption is pivotal in the development of neuroinflammation, peripheral sensitization, and neuropathic pain after peripheral nerve injury. Activation of toll-like receptor 4 and inactivation of Sonic Hedgehog signaling pathways within the endoneurial endothelial cells are key events, resulting in the infiltration of harmful molecules and immunocytes within the nerve parenchyma. However, we showed in a previous study that preemptive inactivation of toll-like receptor 4 signaling or sustained activation of Sonic Hedgehog signaling did not prevent the local alterations observed following peripheral nerve injury, suggesting the implication of another signaling pathway. Using a classical neuropathic pain model, the infraorbital nerve chronic constriction injury (IoN-CCI), we investigated the role of the Wnt/β-catenin pathway in chronic constriction injury-mediated blood–nerve barrier disruption and in its interactions with the toll-like receptor 4 and Sonic Hedgehog pathways. In the IoN-CCI model versus control, mRNA expression levels and/or immunochemical detection of major Wnt/Sonic Hedgehog pathway (Frizzled-7, vascular endothelial-cadherin, Patched-1 and Gli-1) and/or tight junction proteins (Claudin-1, Claudin-5, and Occludin) readouts were assessed. Vascular permeability was assessed by sodium fluorescein extravasation. IoN-CCI induced early alterations in the vascular endothelial-cadherin/β-catenin/Frizzled-7 complex, shown to participate in local blood–nerve barrier disruption via a β-catenin-dependent tight junction protein downregulation. Wnt pathway also mediated a crosstalk between toll-like receptor 4 and Sonic Hedgehog signaling within endoneurial endothelial cells. Nevertheless, preemptive inhibition of Wnt/β-catenin signaling before IoN-CCI could not prevent the downregulation of key Sonic Hedgehog pathway readouts or the disruption of the infraorbital blood–nerve barrier, suggesting that Sonic Hedgehog pathway

Role of the Protein Corona Derived from Human Plasma in Cellular Interactions between Nanoporous Human Serum Albumin Particles and Endothelial Cells.

PubMed

Zyuzin, Mikhail V; Yan, Yan; Hartmann, Raimo; Gause, Katelyn T; Nazarenus, Moritz; Cui, Jiwei; Caruso, Frank; Parak, Wolfgang J

2017-08-16

The presence of a protein corona on various synthetic nanomaterials has been shown to strongly influence how they interact with cells. However, it is unclear if the protein corona also exists on protein particles, and if so, its role in particle-cell interactions. In this study, pure human serum albumin (HSA) particles were fabricated via mesoporous silica particle templating. Our data reveal that various serum proteins adsorbed on the particles, when exposed to human blood plasma, forming a corona. In human umbilical vein endothelial cells (HUVECs), the corona was shown to decrease particle binding to the cell membrane, increase the residence time of particles in early endosomes, and reduce the amount of internalized particles within the first hours of exposure to particles. These findings reveal important information regarding the mechanisms used by vascular endothelial cells to internalize protein-based particulate materials exposed to blood plasma. The ability to control the cellular recognition of these organic particles is expected to aid the advancement of HSA-based materials for intravenous drug delivery.

The Hedgehog gene family of the cnidarian, Nematostella vectensis, and implications for understanding metazoan Hedgehog pathway evolution.

PubMed

Matus, David Q; Magie, Craig R; Pang, Kevin; Martindale, Mark Q; Thomsen, Gerald H

2008-01-15

Hedgehog signaling is an important component of cell-cell communication during bilaterian development, and abnormal Hedgehog signaling contributes to disease and birth defects. Hedgehog genes are composed of a ligand ("hedge") domain and an autocatalytic intein ("hog") domain. Hedgehog (hh) ligands bind to a conserved set of receptors and activate downstream signal transduction pathways terminating with Gli/Ci transcription factors. We have identified five intein-containing genes in the anthozoan cnidarian Nematostella vectensis, two of which (NvHh1 and NvHh2) contain definitive hedgehog ligand domains, suggesting that to date, cnidarians are the earliest branching metazoan phylum to possess definitive Hh orthologs. Expression analysis of NvHh1 and NvHh2, the receptor NvPatched, and a downstream transcription factor NvGli (a Gli3/Ci ortholog) indicate that these genes may have conserved roles in planar and trans-epithelial signaling during gut and germline development, while the three remaining intein-containing genes (NvHint1,2,3) are expressed in a cell-type-specific manner in putative neural precursors. Metazoan intein-containing genes that lack a hh ligand domain have previously only been identified within nematodes. However, we have identified intein-containing genes from both Nematostella and in two newly annotated lophotrochozoan genomes. Phylogenetic analyses suggest that while nematode inteins may be derived from an ancestral true hedgehog gene, the newly identified cnidarian and lophotrochozoan inteins may be orthologous, suggesting that both true hedgehog and hint genes may have been present in the cnidarian-bilaterian ancestor. Genomic surveys of N. vectensis suggest that most of the components of both protostome and deuterostome Hh signaling pathways are present in anthozoans and that some appear to have been lost in ecdysozoan lineages. Cnidarians possess many bilaterian cell-cell signaling pathways (Wnt, TGFbeta, FGF, and Hh) that appear to act in

The Hedgehog gene family of the cnidarian, Nematostella vectensis, and implications for understanding metazoan Hedgehog pathway evolution

PubMed Central

Matus, David Q.; Magie, Craig; Pang, Kevin; Martindale, Mark Q; Thomsen, Gerald H.

2008-01-01

Hedgehog signaling is an important component of cell-cell communication during bilaterian development, and abnormal Hedgehog signaling contributes to disease and birth defects. Hedgehog genes are composed of a ligand (“hedge”) domain and an autocatalytic intein (“hog”) domain. Hedgehog (hh) ligands bind to a conserved set of receptors and activate downstream signal transduction pathways terminating with Gli/Ci transcription factors. We have identified five intein-containing genes in the anthozoan cnidarian Nematostella vectensis, two of which (NvHh1 and NvHh2) contain definitive hedgehog ligand domains, suggesting that to date, cnidarians are the earliest branching metazoan phylum to possess definitive Hh orthologs. Expression analysis of NvHh1 and NvHh2, the receptor NvPatched and a downstream transcription factor NvGli (a Gli3/Ci ortholog) indicate that these genes may have conserved roles in planar and trans-epithelial signaling during gut and germline development, while the three remaining intein-containing genes (NvHint1,2,3) are expressed in a cell-type specific manner in putative neural precursors. Metazoan intein-containing genes that lack a ligand domain have previously only been identified within nematodes. However, phylogenetic analyses suggest that these nematode inteins may be derived from an ancestral nematode true hedgehog gene, and that the non-bilaterian intein-containing genes identified here may represent an ancestral state prior to the domain swapping events that resulted in the formation of true hedgehog genes in the cnidarian-bilaterian ancestor. Genomic surveys of N. vectensis suggest that most of the components of both protostome and deuterostome Hh signaling pathways are present in anthozoans and that some appear to have been lost in ecdysozoan lineages. Cnidarians possess many bilaterian cell-cell signaling pathways (Wnt, TGFß, FGF and Hh) that appear to act in concert to pattern tissues along the oral-aboral axis of the polyp

Predicting Physical Interactions between Protein Complexes*

PubMed Central

Clancy, Trevor; Rødland, Einar Andreas; Nygard, Ståle; Hovig, Eivind

2013-01-01

Protein complexes enact most biochemical functions in the cell. Dynamic interactions between protein complexes are frequent in many cellular processes. As they are often of a transient nature, they may be difficult to detect using current genome-wide screens. Here, we describe a method to computationally predict physical interactions between protein complexes, applied to both humans and yeast. We integrated manually curated protein complexes and physical protein interaction networks, and we designed a statistical method to identify pairs of protein complexes where the number of protein interactions between a complex pair is due to an actual physical interaction between the complexes. An evaluation against manually curated physical complex-complex interactions in yeast revealed that 50% of these interactions could be predicted in this manner. A community network analysis of the highest scoring pairs revealed a biologically sensible organization of physical complex-complex interactions in the cell. Such analyses of proteomes may serve as a guide to the discovery of novel functional cellular relationships. PMID:23438732

Quality control methodology for high-throughput protein-protein interaction screening.

PubMed

Vazquez, Alexei; Rual, Jean-François; Venkatesan, Kavitha

2011-01-01

Protein-protein interactions are key to many aspects of the cell, including its cytoskeletal structure, the signaling processes in which it is involved, or its metabolism. Failure to form protein complexes or signaling cascades may sometimes translate into pathologic conditions such as cancer or neurodegenerative diseases. The set of all protein interactions between the proteins encoded by an organism constitutes its protein interaction network, representing a scaffold for biological function. Knowing the protein interaction network of an organism, combined with other sources of biological information, can unravel fundamental biological circuits and may help better understand the molecular basics of human diseases. The protein interaction network of an organism can be mapped by combining data obtained from both low-throughput screens, i.e., "one gene at a time" experiments and high-throughput screens, i.e., screens designed to interrogate large sets of proteins at once. In either case, quality controls are required to deal with the inherent imperfect nature of experimental assays. In this chapter, we discuss experimental and statistical methodologies to quantify error rates in high-throughput protein-protein interactions screens.

Sonic Hedgehog Initiates Cochlear Hair Cell Regeneration through Downregulation of Retinoblastoma Protein

PubMed Central

Lu, Na; Chen, Yan; Wang, Zhengmin; Chen, Guoling; Lin, Qin; Chen, Zheng-Yi; Li, Huawei

2013-01-01

Cell cycle re-entry by cochlear supporting cells and/or hair cells is considered one of the best approaches for restoring hearing loss as a result of hair cell damage. To identify mechanisms that can be modulated to initiate cell cycle re-entry and hair cell regeneration, we studied the effect of activating the sonic hedgehog (Shh) pathway. We show that Shh signaling in postnatal rat cochleae damaged by neomycin leads to renewed proliferation of supporting cells and hair cells. Further, proliferating supporting cells are likely to transdifferentiate into hair cells. Shh treatment leads to inhibition of retinoblastoma protein (pRb) by increasing phosphorylated pRb and reducing retinoblastoma gene transcription. This results in upregulation of cyclins B1, D2, and D3, and CDK1. These results suggest that Shh signaling induces cell cycle re-entry in cochlear sensory epithelium and the production of new hair cells, in part by attenuating pRb function. This study provides an additional route to modulate pRb function with important implications in mammalian hair cell regeneration. PMID:23211596

Quantifying protein-protein interactions in high throughput using protein domain microarrays.

PubMed

Kaushansky, Alexis; Allen, John E; Gordus, Andrew; Stiffler, Michael A; Karp, Ethan S; Chang, Bryan H; MacBeath, Gavin

2010-04-01

Protein microarrays provide an efficient way to identify and quantify protein-protein interactions in high throughput. One drawback of this technique is that proteins show a broad range of physicochemical properties and are often difficult to produce recombinantly. To circumvent these problems, we have focused on families of protein interaction domains. Here we provide protocols for constructing microarrays of protein interaction domains in individual wells of 96-well microtiter plates, and for quantifying domain-peptide interactions in high throughput using fluorescently labeled synthetic peptides. As specific examples, we will describe the construction of microarrays of virtually every human Src homology 2 (SH2) and phosphotyrosine binding (PTB) domain, as well as microarrays of mouse PDZ domains, all produced recombinantly in Escherichia coli. For domains that mediate high-affinity interactions, such as SH2 and PTB domains, equilibrium dissociation constants (K(D)s) for their peptide ligands can be measured directly on arrays by obtaining saturation binding curves. For weaker binding domains, such as PDZ domains, arrays are best used to identify candidate interactions, which are then retested and quantified by fluorescence polarization. Overall, protein domain microarrays provide the ability to rapidly identify and quantify protein-ligand interactions with minimal sample consumption. Because entire domain families can be interrogated simultaneously, they provide a powerful way to assess binding selectivity on a proteome-wide scale and provide an unbiased perspective on the connectivity of protein-protein interaction networks.

A human functional protein interaction network and its application to cancer data analysis

PubMed Central

2010-01-01

Background One challenge facing biologists is to tease out useful information from massive data sets for further analysis. A pathway-based analysis may shed light by projecting candidate genes onto protein functional relationship networks. We are building such a pathway-based analysis system. Results We have constructed a protein functional interaction network by extending curated pathways with non-curated sources of information, including protein-protein interactions, gene coexpression, protein domain interaction, Gene Ontology (GO) annotations and text-mined protein interactions, which cover close to 50% of the human proteome. By applying this network to two glioblastoma multiforme (GBM) data sets and projecting cancer candidate genes onto the network, we found that the majority of GBM candidate genes form a cluster and are closer than expected by chance, and the majority of GBM samples have sequence-altered genes in two network modules, one mainly comprising genes whose products are localized in the cytoplasm and plasma membrane, and another comprising gene products in the nucleus. Both modules are highly enriched in known oncogenes, tumor suppressors and genes involved in signal transduction. Similar network patterns were also found in breast, colorectal and pancreatic cancers. Conclusions We have built a highly reliable functional interaction network upon expert-curated pathways and applied this network to the analysis of two genome-wide GBM and several other cancer data sets. The network patterns revealed from our results suggest common mechanisms in the cancer biology. Our system should provide a foundation for a network or pathway-based analysis platform for cancer and other diseases. PMID:20482850

Evaluation of two novel leptospiral proteins for their interaction with human host components.

PubMed

Silva, Lucas P; Fernandes, Luis G V; Vieira, Monica L; de Souza, Gisele O; Heinemann, Marcos B; Vasconcellos, Silvio A; Romero, Eliete C; Nascimento, Ana L T O

2016-07-01

Pathogenic species of the genus Leptospira are the etiological agents of leptospirosis, the most widespread zoonosis. Mechanisms involved in leptospiral pathogenesis are not well understood. By data mining the genome sequences of Leptospira interrogans we have identified two proteins predicted to be surface exposed, LIC10821 and LIC10064. Immunofluorescence and proteinase K assays confirmed that the proteins are exposed. Reactivity of the recombinant proteins with human sera has shown that rLIC10821, but not rLIC10064, is recognized by antibodies in confirmed leptospirosis serum samples, suggesting its expression during infection. The rLIC10821 was able to bind laminin, in a dose-dependent fashion, and was called Lsa37 (leptospiral surface adhesin of 37 kDa). Studies with human plasma components demonstrated that rLIC10821 interacts with plasminogen (PLG) and fibrinogen (Fg). The binding of Lsa37 with PLG generates plasmin when PLG activator was added. Fibrin clotting reduction was observed in a thrombin-catalyzed reaction, when Fg was incubated with Lsa37, suggesting that this protein may interfere in the coagulation cascade during the disease. Although LIC10064 protein is more abundant than the corresponding Lsa37, binding activity with all the components tested was not detected. Thus, Lsa37 is a novel versatile adhesin that may mediate Leptospira-host interactions. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Identification of New Protein Interactions between Dengue Fever Virus and Its Hosts, Human and Mosquito

PubMed Central

Mairiang, Dumrong; Zhang, Huamei; Sodja, Ann; Murali, Thilakam; Suriyaphol, Prapat; Malasit, Prida; Limjindaporn, Thawornchai; Finley, Russell L.

2013-01-01

The four divergent serotypes of dengue virus are the causative agents of dengue fever, dengue hemorrhagic fever and dengue shock syndrome. About two-fifths of the world's population live in areas where dengue is prevalent, and thousands of deaths are caused by the viruses every year. Dengue virus is transmitted from one person to another primarily by the yellow fever mosquito, Aedes aegypti. Recent studies have begun to define how the dengue viral proteins interact with host proteins to mediate viral replication and pathogenesis. A combined analysis of these studies, however, suggests that many virus-host protein interactions remain to be identified, especially for the mosquito host. In this study, we used high-throughput yeast two-hybrid screening to identify mosquito and human proteins that physically interact with dengue proteins. We tested each identified host protein against the proteins from all four serotypes of dengue to identify interactions that are conserved across serotypes. We further confirmed many of the interactions using co-affinity purification assays. As in other large-scale screens, we identified some previously detected interactions and many new ones, moving us closer to a complete host – dengue protein interactome. To help summarize and prioritize the data for further study, we combined our interactions with other published data and identified a subset of the host-dengue interactions that are now supported by multiple forms of evidence. These data should be useful for understanding the interplay between dengue and its hosts and may provide candidates for drug targets and vector control strategies. PMID:23326450

Identification of new protein interactions between dengue fever virus and its hosts, human and mosquito.

PubMed

Mairiang, Dumrong; Zhang, Huamei; Sodja, Ann; Murali, Thilakam; Suriyaphol, Prapat; Malasit, Prida; Limjindaporn, Thawornchai; Finley, Russell L

2013-01-01

The four divergent serotypes of dengue virus are the causative agents of dengue fever, dengue hemorrhagic fever and dengue shock syndrome. About two-fifths of the world's population live in areas where dengue is prevalent, and thousands of deaths are caused by the viruses every year. Dengue virus is transmitted from one person to another primarily by the yellow fever mosquito, Aedes aegypti. Recent studies have begun to define how the dengue viral proteins interact with host proteins to mediate viral replication and pathogenesis. A combined analysis of these studies, however, suggests that many virus-host protein interactions remain to be identified, especially for the mosquito host. In this study, we used high-throughput yeast two-hybrid screening to identify mosquito and human proteins that physically interact with dengue proteins. We tested each identified host protein against the proteins from all four serotypes of dengue to identify interactions that are conserved across serotypes. We further confirmed many of the interactions using co-affinity purification assays. As in other large-scale screens, we identified some previously detected interactions and many new ones, moving us closer to a complete host - dengue protein interactome. To help summarize and prioritize the data for further study, we combined our interactions with other published data and identified a subset of the host-dengue interactions that are now supported by multiple forms of evidence. These data should be useful for understanding the interplay between dengue and its hosts and may provide candidates for drug targets and vector control strategies.

IREB2, CHRNA5, CHRNA3, FAM13A & hedgehog interacting protein genes polymorphisms & risk of chronic obstructive pulmonary disease in Tatar population from Russia.

PubMed

Korytina, Gulnaz Faritovna; Akhmadishina, Leysan Zinurovna; Viktorova, Elena Vitalievna; Kochetova, Olga Vladimirovna; Viktorova, Tatyana Victorovna

2016-12-01

Chronic obstructive pulmonary disease (COPD) is a complex chronic inflammatory disease of the respiratory system affecting primarily distal respiratory pathways and lung parenchyma. This study was aimed at investigating the association of COPD with IREB2, CHRNA5, CHRNA3, FAM13A and hedgehog interacting protein (HHIP) genes in a Tatar population from Russia. Six single nucleotide polymorphisms (SNPs) (rs13180, rs16969968, rs1051730, rs6495309, rs7671167, rs13118928) were genotyped by the real-time polymerase chain reaction in this study (511 COPD patients and 508 controls). Logistic regression was used to detect the association of SNPs and haplotypes of linked loci in different models. Linear regression analyses were performed to estimate the relationship between SNPs and lung function parameters and pack-years. The rs13180 (IREB2), rs16969968 (CHRNA5) and rs1051730 (CHRNA3) were significantly associated with COPD in additive model [Padj =0.00001, odds ratio (OR)=0.64; Padj =0.0001, OR=1.41 and Padj =0.0001, OR=1.47]. The C-G haplotype by rs13180 and rs1051730 was a protective factor for COPD in our population (Padj =0.0005, OR=0.61). These results were confirmed only in smokers. The rs16969968 and rs1051730 were associated with decrease of forced expiratory volume in 1 sec % predicted (Padj =0.005 and Padj =0.0019). Our study showed the association of rs13180, rs16969968 and rs1051730 with COPD and lung function in Tatar population from Russia. Further studies need to be done in other ethnic populations.

[Hedgehog fungi in a dermatological office in Munich : Case reports and review].

PubMed

Kargl, A; Kosse, B; Uhrlaß, S; Koch, D; Krüger, C; Eckert, K; Nenoff, P

2018-02-12

Patient 1: After contact to a central European hedgehog (Erinaceus europaeus), a 50-year-old female with atopy developed erythrosquamous tinea manus on the thumb and thenar eminence of the right hand. The patient had previously been scalded by hot steam at the affected site. The zoophilic dermatophyte Trichophyton erinacei could be cultured from the hedgehog as well as from scrapings from the woman's skin. Antifungal treatment of the hedgehog was initiated using 2 weekly cycles of itraconazole solution (0.1 ml/kg body weight, BW). In addition, every other day enilconazole solution was used for topical treatment. The patient was treated with ciclopirox olamine cream and oral terbinafine 250 mg daily for 2 weeks, which led to healing of the Tinea manus .Patient 2: An 18-year-old woman presented for emergency consultation with rimmed, papulous, vesicular and erosive crusted skin lesions of the index finger, and an erythematous dry scaling round lesion on the thigh. The patient worked at an animal care facility, specifically caring for hedgehogs. One of the hedgehogs suffered from a substantial loss of spines. Fungal cultures from skin scrapings of both lesions yielded T. erinacei. Treatment with ciclopirox olamine cream and oral terbinafine 250 mg for 14 days was initiated which led to healing of the lesions. Identification of all three T. erinacei isolates from both patients and from the hedgehog was confirmed by sequencing of the internal transcribed spacer (ITS) region of the ribosomal DNA, and of the translation elongation factor (TEF)-1-alpha gene. Using ITS sequencing discrimination between T. erinacei strains from European and from African hedgehogs is possible. T. erinacei should be considered a so-called emerging pathogen. In Germany the zoophilic dermatophyte T. erinacei should be taken into account as causative agent of dermatomycoses in humans after contact to hedgehogs.

Intraflagellar transport protein 122 antagonizes Sonic Hedgehog signaling and controls ciliary localization of pathway components.

PubMed

Qin, Jian; Lin, Yulian; Norman, Ryan X; Ko, Hyuk W; Eggenschwiler, Jonathan T

2011-01-25

Primary cilia are required for proper Sonic Hedgehog (Shh) signaling in mammals. However, their role in the signal transduction process remains unclear. We have identified sister of open brain (sopb), a null allele of mouse Intraflagellar transport protein 122 (Ift122). IFT122 negatively regulates the Shh pathway in the cilium at a step downstream of the Shh ligand and the transmembrane protein Smoothened, but upstream of the Gli2 transcription factor. Ift122(sopb) mutants generate primary cilia, but they show features of defective retrograde intraflagellar transport. IFT122 controls the ciliary localization of Shh pathway regulators in different ways. Disruption of IFT122 leads to accumulation of Gli2 and Gli3 at cilia tips while blocking the ciliary localization of the antagonist TULP3. Suppressor of Fused and Smoothened localize to the cilium through an IFT122-independent mechanism. We propose that the balance between positive and negative regulators of the Shh pathway at the cilium tip controls the output of the pathway and that Shh signaling regulates this balance through intraflagellar transport.

Myxoma of the penis in an African pygmy hedgehog (Atelerix albiventris).

PubMed

Takami, Yoshinori; Yasuda, Namie; Une, Yumi

2017-01-20

A penile tumor (4 × 2.5 × 1 cm) was surgically removed from an African pygmy hedgehog (Atelerix albiventris) aged 3 years and 5 months. The tumor was continuous with the dorsal fascia of the penile head. Histopathologically, tumor cells were pleomorphic (oval-, short spindle- and star-shaped cells) with low cell density. Abundant edematous stroma was weakly positive for Alcian blue staining and positive for colloidal iron reaction. Tumor cells displayed no cellular atypia or karyokinesis. Tumor cell cytoplasm was positive for vimentin antibody, while cytoplasm and nuclei were positive for S-100 protein antibody. Tumor cell ultrastructure matched that of fibroblasts, and the rough endoplasmic reticulum was enlarged. The tumor was diagnosed as myxoma. This represents the first report of myxoma in a hedgehog.

Hedgehog signaling regulates segment formation in the annelid Platynereis.

PubMed

Dray, Nicolas; Tessmar-Raible, Kristin; Le Gouar, Martine; Vibert, Laura; Christodoulou, Foteini; Schipany, Katharina; Guillou, Aurélien; Zantke, Juliane; Snyman, Heidi; Béhague, Julien; Vervoort, Michel; Arendt, Detlev; Balavoine, Guillaume

2010-07-16

Annelids and arthropods share a similar segmented organization of the body whose evolutionary origin remains unclear. The Hedgehog signaling pathway, prominent in arthropod embryonic segment patterning, has not been shown to have a similar function outside arthropods. We show that the ligand Hedgehog, the receptor Patched, and the transcription factor Gli are all expressed in striped patterns before the morphological appearance of segments in the annelid Platynereis dumerilii. Treatments with small molecules antagonistic to Hedgehog signaling disrupt segment formation. Platynereis Hedgehog is not necessary to establish early segment patterns but is required to maintain them. The molecular similarity of segment patterning functions of the Hedgehog pathway in an annelid and in arthropods supports a common origin of segmentation in protostomes.

Exploring the Human-Nipah Virus Protein-Protein Interactome

PubMed Central

Vera-Velasco, Natalia M.; Mingarro, Ismael

2017-01-01

ABSTRACT Nipah virus is an emerging, highly pathogenic, zoonotic virus of the Paramyxoviridae family. Human transmission occurs by close contact with infected animals, the consumption of contaminated food, or, occasionally, via other infected individuals. Currently, we lack therapeutic or prophylactic treatments for Nipah virus. To develop these agents we must now improve our understanding of the host-virus interactions that underpin a productive infection. This aim led us to perform the present work, in which we identified 101 human-Nipah virus protein-protein interactions (PPIs), most of which (88) are novel. This data set provides a comprehensive view of the host complexes that are manipulated by viral proteins. Host targets include the PRP19 complex and the microRNA (miRNA) processing machinery. Furthermore, we explored the biologic consequences of the interaction with the PRP19 complex and found that the Nipah virus W protein is capable of altering p53 control and gene expression. We anticipate that these data will help in guiding the development of novel interventional strategies to counter this emerging viral threat. IMPORTANCE Nipah virus is a recently discovered virus that infects a wide range of mammals, including humans. Since its discovery there have been yearly outbreaks, and in some of them the mortality rate has reached 100% of the confirmed cases. However, the study of Nipah virus has been largely neglected, and currently we lack treatments for this infection. To develop these agents we must now improve our understanding of the host-virus interactions that underpin a productive infection. In the present work, we identified 101 human-Nipah virus protein-protein interactions using an affinity purification approach coupled with mass spectrometry. Additionally, we explored the cellular consequences of some of these interactions. Globally, this data set offers a comprehensive and detailed view of the host machinery's contribution to the Nipah virus

Exploring the Human-Nipah Virus Protein-Protein Interactome.

PubMed

Martinez-Gil, Luis; Vera-Velasco, Natalia M; Mingarro, Ismael

2017-12-01

Nipah virus is an emerging, highly pathogenic, zoonotic virus of the Paramyxoviridae family. Human transmission occurs by close contact with infected animals, the consumption of contaminated food, or, occasionally, via other infected individuals. Currently, we lack therapeutic or prophylactic treatments for Nipah virus. To develop these agents we must now improve our understanding of the host-virus interactions that underpin a productive infection. This aim led us to perform the present work, in which we identified 101 human-Nipah virus protein-protein interactions (PPIs), most of which (88) are novel. This data set provides a comprehensive view of the host complexes that are manipulated by viral proteins. Host targets include the PRP19 complex and the microRNA (miRNA) processing machinery. Furthermore, we explored the biologic consequences of the interaction with the PRP19 complex and found that the Nipah virus W protein is capable of altering p53 control and gene expression. We anticipate that these data will help in guiding the development of novel interventional strategies to counter this emerging viral threat. IMPORTANCE Nipah virus is a recently discovered virus that infects a wide range of mammals, including humans. Since its discovery there have been yearly outbreaks, and in some of them the mortality rate has reached 100% of the confirmed cases. However, the study of Nipah virus has been largely neglected, and currently we lack treatments for this infection. To develop these agents we must now improve our understanding of the host-virus interactions that underpin a productive infection. In the present work, we identified 101 human-Nipah virus protein-protein interactions using an affinity purification approach coupled with mass spectrometry. Additionally, we explored the cellular consequences of some of these interactions. Globally, this data set offers a comprehensive and detailed view of the host machinery's contribution to the Nipah virus's life cycle

Neuropilins are positive regulators of Hedgehog signal transduction

PubMed Central

Hillman, R. Tyler; Feng, Brian Y.; Ni, Jun; Woo, Wei-Meng; Milenkovic, Ljiljana; Hayden Gephart, Melanie G.; Teruel, Mary N.; Oro, Anthony E.; Chen, James K.; Scott, Matthew P.

2011-01-01

The Hedgehog (Hh) pathway is essential for vertebrate embryogenesis, and excessive Hh target gene activation can cause cancer in humans. Here we show that Neuropilin 1 (Nrp1) and Nrp2, transmembrane proteins with roles in axon guidance and vascular endothelial growth factor (VEGF) signaling, are important positive regulators of Hh signal transduction. Nrps are expressed at times and locations of active Hh signal transduction during mouse development. Using cell lines lacking key Hh pathway components, we show that Nrps mediate Hh transduction between activated Smoothened (Smo) protein and the negative regulator Suppressor of Fused (SuFu). Nrp1 transcription is induced by Hh signaling, and Nrp1 overexpression increases maximal Hh target gene activation, indicating the existence of a positive feedback circuit. The regulation of Hh signal transduction by Nrps is conserved between mammals and bony fish, as we show that morpholinos targeting the Nrp zebrafish ortholog nrp1a produce a specific and highly penetrant Hh pathway loss-of-function phenotype. These findings enhance our knowledge of Hh pathway regulation and provide evidence for a conserved nexus between Nrps and this important developmental signaling system. PMID:22051878

Recombinant Human Sonic Hedgehog Protein Regulates the Expression of ZO-1 and Occludin by Activating Angiopoietin-1 in Stroke Damage

PubMed Central

Chen, Sheng-cai; Huang, Ming; Wang, Yong; Gao, Yuan; Huang, Yan; Wang, Meng-die; Mao, Ling; Hu, Bo

2013-01-01

This study examines the regulating effect of Sonic Hedgehog (Shh) on the permeability of the blood-brain barrier (BBB) in cerebral ischemia. By employing permanent middle cerebral artery occlusion (pMCAO) model, we find that Shh significantly decreases brain edema and preserves BBB permeability. Moreover, Shh increases zonula occludens-1 (ZO-1), occludin and angiopiotetin-1 (Ang-1) expression in the ischemic penumbra. Blockage of Shh with cyclopamine abolishes the effects of Shh on brain edema, BBB permeability and ZO-1, occludin, Ang-1 expression. Primary brain microvessel endothelial cells (BMECs) and astrocytes were pre-treated with Shh, cyclopamine, Ang-1-neutralizing antibody, and subjected to oxygen-glucose deprivation (OGD). Results show that the Ang-1 protein level in the culture medium of Shh-treated astrocytes is significantly higher. Shh also increased ZO-1, occludin and Ang-1 expression in BMECs, while cyclopamine and Ang-1-neutralizing antibody inhibited the effects of Shh on the ZO-1 and occludin expression, respectively. This study suggests that, under ischemic insults, Shh triggers Ang-1 production predominantly in astrocytes, and the secreted Ang-1 acts on BMECs, thereby upregulating ZO-1 and occludin to repair the tight junction and ameliorate the brain edema and BBB leakage. PMID:23894369

Protein-protein interactions and cancer: targeting the central dogma.

PubMed

Garner, Amanda L; Janda, Kim D

2011-01-01

Between 40,000 and 200,000 protein-protein interactions have been predicted to exist within the human interactome. As these interactions are of a critical nature in many important cellular functions and their dysregulation is causal of disease, the modulation of these binding events has emerged as a leading, yet difficult therapeutic arena. In particular, the targeting of protein-protein interactions relevant to cancer is of fundamental importance as the tumor-promoting function of several aberrantly expressed proteins in the cancerous state is directly resultant of its ability to interact with a protein-binding partner. Of significance, these protein complexes play a crucial role in each of the steps of the central dogma of molecular biology, the fundamental processes of genetic transmission. With the many important discoveries being made regarding the mechanisms of these genetic process, the identification of new chemical probes are needed to better understand and validate the druggability of protein-protein interactions related to the central dogma. In this review, we provide an overview of current small molecule-based protein-protein interaction inhibitors for each stage of the central dogma: transcription, mRNA splicing and translation. Importantly, through our analysis we have uncovered a lack of necessary probes targeting mRNA splicing and translation, thus, opening up the possibility for expansion of these fields.

Regulator of G protein signaling 5 (RGS5) inhibits sonic hedgehog function in mouse cortical neurons.

PubMed

Liu, Chuanliang; Hu, Qiongqiong; Jing, Jia; Zhang, Yun; Jin, Jing; Zhang, Liulei; Mu, Lili; Liu, Yumei; Sun, Bo; Zhang, Tongshuai; Kong, Qingfei; Wang, Guangyou; Wang, Dandan; Zhang, Yao; Liu, Xijun; Zhao, Wei; Wang, Jinghua; Feng, Tao; Li, Hulun

2017-09-01

Regulator of G protein signaling 5 (RGS5) acts as a GTPase-activating protein (GAP) for the Gαi subunit and negatively regulates G protein-coupled receptor signaling. However, its presence and function in postmitotic differentiated primary neurons remains largely uncharacterized. During neural development, sonic hedgehog (Shh) signaling is involved in cell signaling pathways via Gαi activity. In particular, Shh signaling is essential for embryonic neural tube patterning, which has been implicated in neuronal polarization involving neurite outgrowth. Here, we examined whether RGS5 regulates Shh signaling in neurons. RGS5 transcripts were found to be expressed in cortical neurons and their expression gradually declined in a time-dependent manner in culture system. When an adenovirus expressing RGS5 was introduced into an in vitro cell culture model of cortical neurons, RGS5 overexpression significantly reduced neurite outgrowth and FM4-64 uptake, while cAMP-PKA signaling was also affected. These findings suggest that RGS5 inhibits Shh function during neurite outgrowth and the presynaptic terminals of primary cortical neurons mature via modulation of cAMP. Copyright © 2017 Elsevier Inc. All rights reserved.

Topology and weights in a protein domain interaction network--a novel way to predict protein interactions.

PubMed

Wuchty, Stefan

2006-05-23

While the analysis of unweighted biological webs as diverse as genetic, protein and metabolic networks allowed spectacular insights in the inner workings of a cell, biological networks are not only determined by their static grid of links. In fact, we expect that the heterogeneity in the utilization of connections has a major impact on the organization of cellular activities as well. We consider a web of interactions between protein domains of the Protein Family database (PFAM), which are weighted by a probability score. We apply metrics that combine the static layout and the weights of the underlying interactions. We observe that unweighted measures as well as their weighted counterparts largely share the same trends in the underlying domain interaction network. However, we only find weak signals that weights and the static grid of interactions are connected entities. Therefore assuming that a protein interaction is governed by a single domain interaction, we observe strong and significant correlations of the highest scoring domain interaction and the confidence of protein interactions in the underlying interactions of yeast and fly. Modeling an interaction between proteins if we find a high scoring protein domain interaction we obtain 1, 428 protein interactions among 361 proteins in the human malaria parasite Plasmodium falciparum. Assessing their quality by a logistic regression method we observe that increasing confidence of predicted interactions is accompanied by high scoring domain interactions and elevated levels of functional similarity and evolutionary conservation. Our results indicate that probability scores are randomly distributed, allowing to treat static grid and weights of domain interactions as separate entities. In particular, these finding confirms earlier observations that a protein interaction is a matter of a single interaction event on domain level. As an immediate application, we show a simple way to predict potential protein interactions

Bacterial co-expression of human Tau protein with protein kinase A and 14-3-3 for studies of 14-3-3/phospho-Tau interaction

PubMed Central

Tugaeva, Kristina V.; Tsvetkov, Philipp O.

2017-01-01

Abundant regulatory 14-3-3 proteins have an extremely wide interactome and coordinate multiple cellular events via interaction with specifically phosphorylated partner proteins. Notwithstanding the key role of 14-3-3/phosphotarget interactions in many physiological and pathological processes, they are dramatically underexplored. Here, we focused on the 14-3-3 interaction with human Tau protein associated with the development of several neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. Among many known phosphorylation sites within Tau, protein kinase A (PKA) phosphorylates several key residues of Tau and induces its tight interaction with 14-3-3 proteins. However, the stoichiometry and mechanism of 14-3-3 interaction with phosphorylated Tau (pTau) are not clearly elucidated. In this work, we describe a simple bacterial co-expression system aimed to facilitate biochemical and structural studies on the 14-3-3/pTau interaction. We show that dual co-expression of human fetal Tau with PKA in Escherichia coli results in multisite Tau phosphorylation including also naturally occurring sites which were not previously considered in the context of 14-3-3 binding. Tau protein co-expressed with PKA displays tight functional interaction with 14-3-3 isoforms of a different type. Upon triple co-expression with 14-3-3 and PKA, Tau protein could be co-purified with 14-3-3 and demonstrates complex which is similar to that formed in vitro between individual 14-3-3 and pTau obtained from dual co-expression. Although used in this study for the specific case of the previously known 14-3-3/pTau interaction, our co-expression system may be useful to study of other selected 14-3-3/phosphotarget interactions and for validations of 14-3-3 complexes identified by other methods. PMID:28575131

A Viral-Human Interactome Based on Structural Motif-Domain Interactions Captures the Human Infectome

PubMed Central

Guo, Xianwu; Rodríguez-Pérez, Mario A.

2013-01-01

Protein interactions between a pathogen and its host are fundamental in the establishment of the pathogen and underline the infection mechanism. In the present work, we developed a single predictive model for building a host-viral interactome based on the identification of structural descriptors from motif-domain interactions of protein complexes deposited in the Protein Data Bank (PDB). The structural descriptors were used for searching, in a database of protein sequences of human and five clinically important viruses; therefore, viral and human proteins sharing a descriptor were predicted as interacting proteins. The analysis of the host-viral interactome allowed to identify a set of new interactions that further explain molecular mechanism associated with viral infections and showed that it was able to capture human proteins already associated to viral infections (human infectome) and non-infectious diseases (human diseasome). The analysis of human proteins targeted by viral proteins in the context of a human interactome showed that their neighbors are enriched in proteins reported with differential expression under infection and disease conditions. It is expected that the findings of this work will contribute to the development of systems biology for infectious diseases, and help guide the rational identification and prioritization of novel drug targets. PMID:23951184

Suppression of hedgehog signaling regulates hepatic stellate cell activation and collagen secretion.

PubMed

Li, Tao; Leng, Xi-Sheng; Zhu, Ji-Ye; Wang, Gang

2015-01-01

Hepatic stellate cells (HSCs) play an important role in liver fibrosis. This study investigates the expression of hedgehog in HSC and the role of hedgehog signaling on activation and collagen secretion of HSC. Liver ex vivo perfusion with collagenase IV and density gradient centrifugation were used to isolate HSC. Expression of hedgehog signaling components Ihh, Smo, Ptc, Gli2 and Gli3 in HSC were detected by RT-PCR. Hedgehog siRNA vectors targeting Ihh, Smo and Gli2 were constructed and transfected into HSC respectively. Suppression of hedgehog signaling were detected by SYBR Green fluorescence quantitative RT-PCR. Effects of hedgehog signaling inhibition on HSC activation and collagen I secretion were analyzed. Hedgehog signaling components Ihh, Smo, Ptc, Gli2 and Gli3 were expressed in HSC. siRNA vectors targeting Ihh, Smo and Gli2 were successfully constructed and decreased target gene expression. Suppression of hedgehog signaling significantly decreased the expression of α-SMA in HSC (P<0.01). Collagen type I secretion of HSC were also significantly decreased (P<0.01). In summary, HSC activation and collagen secretion can be regulated by hedgehog signaling. Hedgehog may play a role in the pathogenesis of liver fibrosis.

The crystal structure of a bacterial Sufu-like protein defines a novel group of bacterial proteins that are similar to the N-terminal domain of human Sufu

PubMed Central

Das, Debanu; Finn, Robert D; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L; Bakolitsa, Constantina; Cai, Xiaohui; Carlton, Dennis; Chen, Connie; Chiu, Hsiu-Ju; Chiu, Michelle; Clayton, Thomas; Deller, Marc C; Duan, Lian; Ellrott, Kyle; Farr, Carol L; Feuerhelm, Julie; Grant, Joanna C; Grzechnik, Anna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K; Klock, Heath E; Knuth, Mark W; Kozbial, Piotr; Sri Krishna, S; Kumar, Abhinav; Lam, Winnie W; Marciano, David; Miller, Mitchell D; Morse, Andrew T; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Puckett, Christina; Reyes, Ron; Tien, Henry J; Trame, Christine B; van den Bedem, Henry; Weekes, Dana; Wooten, Tiffany; Xu, Qingping; Yeh, Andrew; Zhou, Jiadong; Hodgson, Keith O; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wilson, Ian A

2010-01-01

Sufu (Suppressor of Fused), a two-domain protein, plays a critical role in regulating Hedgehog signaling and is conserved from flies to humans. A few bacterial Sufu-like proteins have previously been identified based on sequence similarity to the N-terminal domain of eukaryotic Sufu proteins, but none have been structurally or biochemically characterized and their function in bacteria is unknown. We have determined the crystal structure of a more distantly related Sufu-like homolog, NGO1391 from Neisseria gonorrhoeae, at 1.4 Å resolution, which provides the first biophysical characterization of a bacterial Sufu-like protein. The structure revealed a striking similarity to the N-terminal domain of human Sufu (r.m.s.d. of 2.6 Å over 93% of the NGO1391 protein), despite an extremely low sequence identity of ∼15%. Subsequent sequence analysis revealed that NGO1391 defines a new subset of smaller, Sufu-like proteins that are present in ∼200 bacterial species and has resulted in expansion of the SUFU (PF05076) family in Pfam. PMID:20836087

Hedgehog inhibition promotes a switch from Type II to Type I cell death receptor signaling in cancer cells.

PubMed

Kurita, Satoshi; Mott, Justin L; Cazanave, Sophie C; Fingas, Christian D; Guicciardi, Maria E; Bronk, Steve F; Roberts, Lewis R; Fernandez-Zapico, Martin E; Gores, Gregory J

2011-03-31

TRAIL is a promising therapeutic agent for human malignancies. TRAIL often requires mitochondrial dysfunction, referred to as the Type II death receptor pathway, to promote cytotoxicity. However, numerous malignant cells are TRAIL resistant due to inhibition of this mitochondrial pathway. Using cholangiocarcinoma cells as a model of TRAIL resistance, we found that Hedgehog signaling blockade sensitized these cancer cells to TRAIL cytotoxicity independent of mitochondrial dysfunction, referred to as Type I death receptor signaling. This switch in TRAIL requirement from Type II to Type I death receptor signaling was demonstrated by the lack of functional dependence on Bid/Bim and Bax/Bak, proapoptotic components of the mitochondrial pathway. Hedgehog signaling modulated expression of X-linked inhibitor of apoptosis (XIAP), which serves to repress the Type I death receptor pathway. siRNA targeted knockdown of XIAP mimics sensitization to mitochondria-independent TRAIL killing achieved by Hedgehog inhibition. Regulation of XIAP expression by Hedgehog signaling is mediated by the glioma-associated oncogene 2 (GLI2), a downstream transcription factor of Hedgehog. In conclusion, these data provide additional mechanisms modulating cell death by TRAIL and suggest Hedgehog inhibition as a therapeutic approach for TRAIL-resistant neoplasms.

Phosphorylation of human tristetraprolin in response to its interaction with the Cbl interacting protein CIN85.

PubMed

Kedar, Vishram P; Darby, Martyn K; Williams, Jason G; Blackshear, Perry J

2010-03-08

Tristetraprolin (TTP) is the prototype member of a family of CCCH tandem zinc finger proteins and is considered to be an anti-inflammatory protein in mammals. TTP plays a critical role in the decay of tumor necrosis factor alpha (TNF) mRNA, among others, by binding AU-rich RNA elements in the 3'-untranslated regions of this transcript and promoting its deadenylation and degradation. We used yeast two-hybrid analysis to identify potential protein binding partners for human TTP (hTTP). Various regions of hTTP recovered 31 proteins that fell into 12 categories based on sequence similarities. Among these, the interactions between hTTP and CIN85, cytoplasmic poly (A) binding protein (PABP), nucleolin and heat shock protein 70 were confirmed by co-immunoprecipitation experiments. CIN85 and hTTP co-localized in the cytoplasm of cells as determined by confocal microscopy. CIN85 contains three SH3 domains that specifically bind a unique proline-arginine motif (PXXXPR) found in several CIN85 effectors. We found that the SH3 domains of CIN85 bound to a PXXXPR motif located near the C-terminus of hTTP. Co-expression of CIN85 with hTTP resulted in the increased phosphorylation of hTTP at serine residues in positions 66 and 93, possibly due in part to the demonstrated association of mitogen-activated protein kinase kinase kinase 4 (MEKK4) to both proteins. The presence of CIN85 did not appear to alter hTTP's binding to RNA probes or its stimulated breakdown of TNF mRNA. These studies describe interactions between hTTP and nucleolin, cytoplasmic PABP, heat shock protein 70 and CIN85; these interactions were initially discovered by two-hybrid analysis, and confirmed by co-immunoprecipitation. We found that CIN85 binding to a C-terminal motif within hTTP led to the increased phosphorylation of hTTP, possibly through enhanced association with MEKK4. The functional consequences to each of the members of this putative complex remain to be determined.

Controllability analysis of the directed human protein interaction network identifies disease genes and drug targets

PubMed Central

Vinayagam, Arunachalam; Gibson, Travis E.; Lee, Ho-Joon; Yilmazel, Bahar; Roesel, Charles; Hu, Yanhui; Kwon, Young; Sharma, Amitabh; Liu, Yang-Yu; Perrimon, Norbert; Barabási, Albert-László

2016-01-01

The protein–protein interaction (PPI) network is crucial for cellular information processing and decision-making. With suitable inputs, PPI networks drive the cells to diverse functional outcomes such as cell proliferation or cell death. Here, we characterize the structural controllability of a large directed human PPI network comprising 6,339 proteins and 34,813 interactions. This network allows us to classify proteins as “indispensable,” “neutral,” or “dispensable,” which correlates to increasing, no effect, or decreasing the number of driver nodes in the network upon removal of that protein. We find that 21% of the proteins in the PPI network are indispensable. Interestingly, these indispensable proteins are the primary targets of disease-causing mutations, human viruses, and drugs, suggesting that altering a network’s control property is critical for the transition between healthy and disease states. Furthermore, analyzing copy number alterations data from 1,547 cancer patients reveals that 56 genes that are frequently amplified or deleted in nine different cancers are indispensable. Among the 56 genes, 46 of them have not been previously associated with cancer. This suggests that controllability analysis is very useful in identifying novel disease genes and potential drug targets. PMID:27091990

Myxoma of the penis in an African pygmy hedgehog (Atelerix albiventris)

PubMed Central

TAKAMI, Yoshinori; YASUDA, Namie; UNE, Yumi

2016-01-01

A penile tumor (4 × 2.5 × 1 cm) was surgically removed from an African pygmy hedgehog (Atelerix albiventris) aged 3 years and 5 months. The tumor was continuous with the dorsal fascia of the penile head. Histopathologically, tumor cells were pleomorphic (oval-, short spindle- and star-shaped cells) with low cell density. Abundant edematous stroma was weakly positive for Alcian blue staining and positive for colloidal iron reaction. Tumor cells displayed no cellular atypia or karyokinesis. Tumor cell cytoplasm was positive for vimentin antibody, while cytoplasm and nuclei were positive for S-100 protein antibody. Tumor cell ultrastructure matched that of fibroblasts, and the rough endoplasmic reticulum was enlarged. The tumor was diagnosed as myxoma. This represents the first report of myxoma in a hedgehog. PMID:27784859

Hedgehog signaling and laminin play unique and synergistic roles in muscle development.

PubMed

Peterson, Matthew T; Henry, Clarissa A

2010-03-01

Hedgehog (Hh) signaling and laminin-111, a basement membrane protein, are required for early muscle development. Hh signaling specifies different populations of muscle fibers and laminin-111 is critical for early muscle morphogenesis. However, additional requirements for Hh signaling and laminin during later phases of muscle development are not known. Furthermore, interactions between Hh signaling and laminin in this context are unknown. We used laminin gamma1 mutant zebrafish and cyclopamine to block Hh signal transduction separately and in combination to investigate their functions and interactions. We found that both Hh signaling and laminin are required for normal myosin chain expression. In addition, Hh signaling and laminin act synergistically during fast-twitch fiber elongation: fast muscle cells do not elongate in embryos deficient for both Hh signaling and laminin. Finally, we present evidence that suggests that Hh signaling is indirectly required via slow fiber specification for recovery of fast fiber elongation in laminin gamma1 mutant embryos. Copyright (c) 2010 Wiley-Liss, Inc.

Identification of human hnRNP C1/C2 as a dengue virus NS1-interacting protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Noisakran, Sansanee; Medical Molecular Biology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Adulyadejvikrom Building; Sengsai, Suchada

Dengue virus nonstructural protein 1 (NS1) is a key glycoprotein involved in the production of infectious virus and the pathogenesis of dengue diseases. Very little is known how NS1 interacts with host cellular proteins and functions in dengue virus-infected cells. This study aimed at identifying NS1-interacting host cellular proteins in dengue virus-infected cells by employing co-immunoprecipitation, two-dimensional gel electrophoresis, and mass spectrometry. Using lysates of dengue virus-infected human embryonic kidney cells (HEK 293T), immunoprecipitation with an anti-NS1 monoclonal antibody revealed eight isoforms of dengue virus NS1 and a 40-kDa protein, which was subsequently identified by quadrupole time-of-flight tandem mass spectrometrymore » (Q-TOF MS/MS) as human heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Further investigation by co-immunoprecipitation and co-localization confirmed the association of hnRNP C1/C2 and dengue virus NS1 proteins in dengue virus-infected cells. Their interaction may have implications in virus replication and/or cellular responses favorable to survival of the virus in host cells.« less

Mapping the Interactions of Dengue Virus NS1 Protein with Human Liver Proteins Using a Yeast Two-Hybrid System: Identification of C1q as an Interacting Partner

PubMed Central

Allonso, Diego; Nogueira, Mauricio L.; Mohana-Borges, Ronaldo

2013-01-01

Dengue constitutes a global health concern. The clinical manifestation of this disease varies from mild febrile illness to severe hemorrhage and/or fatal hypovolemic shock. Flavivirus nonstructural protein 1 (NS1) is a secreted glycoprotein that is displayed on the surface of infected cells but is absent in viral particles. NS1 accumulates at high levels in the plasma of dengue virus (DENV)-infected patients, and previous reports highlight its involvement in immune evasion, dengue severity, liver dysfunction and pathogenesis. In the present study, we performed a yeast two-hybrid screen to search for DENV2 NS1-interacting partners using a human liver cDNA library. We identified fifty genes, including human complement component 1 (C1q), which was confirmed by coimmunoprecipitation, ELISA and immunofluorescence assays, revealing for the first time the direct binding of this protein to NS1. Furthermore, the majority of the identified genes encode proteins that are secreted into the plasma of patients, and most of these proteins are classified as acute-phase proteins (APPs), such as plasminogen, haptoglobin, hemopexin, α-2-HS-glycoprotein, retinol binding protein 4, transferrin, and C4. The results presented here confirm the direct interaction of DENV NS1 with a key protein of the complement system and suggest a role for this complement protein in the pathogenesis of DENV infection. PMID:23516407

Intestinal plasmacytoma in an African hedgehog.

PubMed

Ramos-Vara, J A; Miller, M A; Craft, D

1998-04-01

A 3-yr-old male African hedgehog (Atelerix albiventris) had anorexia and weight loss for 1 wk before its death. The colon and mesocolon were diffusely infiltrated by a neoplastic proliferation of round cells with plasmacytoid features. A diagnosis of intestinal plasmacytoma was made and confirmed by electron microscopy. No other organs appeared to be affected. This is the first description of intestinal plasmacytoma in a hedgehog.

A credit-card library approach for disrupting protein-protein interactions.

PubMed

Xu, Yang; Shi, Jin; Yamamoto, Noboru; Moss, Jason A; Vogt, Peter K; Janda, Kim D

2006-04-15

Protein-protein interfaces are prominent in many therapeutically important targets. Using small organic molecules to disrupt protein-protein interactions is a current challenge in chemical biology. An important example of protein-protein interactions is provided by the Myc protein, which is frequently deregulated in human cancers. Myc belongs to the family of basic helix-loop-helix leucine zipper (bHLH-ZIP) transcription factors. It is biologically active only as heterodimer with the bHLH-ZIP protein Max. Herein, we report a new strategy for the disruption of protein-protein interactions that has been corroborated through the design and synthesis of a small parallel library composed of 'credit-card' compounds. These compounds are derived from a planar, aromatic scaffold and functionalized with four points of diversity. From a 285 membered library, several hits were obtained that disrupted the c-Myc-Max interaction and cellular functions of c-Myc. The IC50 values determined for this small focused library for the disruption of Myc-Max dimerization are quite potent, especially since small molecule antagonists of protein-protein interactions are notoriously difficult to find. Furthermore, several of the compounds were active at the cellular level as shown by their biological effects on Myc action in chicken embryo fibroblast assays. In light of our findings, this approach is considered a valuable addition to the armamentarium of new molecules being developed to interact with protein-protein interfaces. Finally, this strategy for disrupting protein-protein interactions should prove applicable to other families of proteins.

Predicting disease-related proteins based on clique backbone in protein-protein interaction network.

PubMed

Yang, Lei; Zhao, Xudong; Tang, Xianglong

2014-01-01

Network biology integrates different kinds of data, including physical or functional networks and disease gene sets, to interpret human disease. A clique (maximal complete subgraph) in a protein-protein interaction network is a topological module and possesses inherently biological significance. A disease-related clique possibly associates with complex diseases. Fully identifying disease components in a clique is conductive to uncovering disease mechanisms. This paper proposes an approach of predicting disease proteins based on cliques in a protein-protein interaction network. To tolerate false positive and negative interactions in protein networks, extending cliques and scoring predicted disease proteins with gene ontology terms are introduced to the clique-based method. Precisions of predicted disease proteins are verified by disease phenotypes and steadily keep to more than 95%. The predicted disease proteins associated with cliques can partly complement mapping between genotype and phenotype, and provide clues for understanding the pathogenesis of serious diseases.

Morphological and immunohistochemical characterization of spontaneous mammary tumours in European hedgehogs (Erinaceus europaeus).

PubMed

Döpke, C; Fehr, M; Thiele, A; Pohlenz, J; Wohlsein, P

2007-07-01

Mammary tumour samples (11 surgical and five post-mortem) from 16 adult European hedgehogs submitted between 1980 and 2004 were examined. Histologically, the tumours were classified as simple tubulo-papillary carcinomas with local invasive growth. In six cases, tumour cell emboli were present in blood vessels or lymphatic vessels, or both. However, metastasis to regional lymph nodes was found only in one hedgehog. Malignant neoplastic epithelial cells were immunolabelled by antibodies specific for various cytokeratins (CKs), including CK1-8, 10, 13-16, 19 and 20. CK expression did not differ from that in normal mammary gland tissue. CK20 was expressed in the mammary tissue of hedgehogs, in contrast to that of dogs and cats; CK7 immunolabelling, however, which commonly occurs in mammary epithelial cells, was negative. CK20 expression, together with the lack of CK7 as determined by a protein-specific antibody, represented an important difference from the CK profile shown by mammary epithelial cells of other mammalian species, including the dog and cat.

Phosphodiesterase 4D acts downstream of Neuropilin to control Hedgehog signal transduction and the growth of medulloblastoma.

PubMed

Ge, Xuecai; Milenkovic, Ljiljana; Suyama, Kaye; Hartl, Tom; Purzner, Teresa; Winans, Amy; Meyer, Tobias; Scott, Matthew P

2015-09-15

Alterations in Hedgehog (Hh) signaling lead to birth defects and cancers including medulloblastoma, the most common pediatric brain tumor. Although inhibitors targeting the membrane protein Smoothened suppress Hh signaling, acquired drug resistance and tumor relapse call for additional therapeutic targets. Here we show that phosphodiesterase 4D (PDE4D) acts downstream of Neuropilins to control Hh transduction and medulloblastoma growth. PDE4D interacts directly with Neuropilins, positive regulators of Hh pathway. The Neuropilin ligand Semaphorin3 enhances this interaction, promoting PDE4D translocation to the plasma membrane and cAMP degradation. The consequent inhibition of protein kinase A (PKA) enhances Hh transduction. In the developing cerebellum, genetic removal of Neuropilins reduces Hh signaling activity and suppresses proliferation of granule neuron precursors. In mouse medulloblastoma allografts, PDE4D inhibitors suppress Hh transduction and inhibit tumor growth. Our findings reveal a new regulatory mechanism of Hh transduction, and highlight PDE4D as a promising target to treat Hh-related tumors.

Reduced primary cilia length and altered Arl13b expression are associated with deregulated chondrocyte Hedgehog signaling in alkaptonuria.

PubMed

Thorpe, Stephen D; Gambassi, Silvia; Thompson, Clare L; Chandrakumar, Charmilie; Santucci, Annalisa; Knight, Martin M

2017-09-01

Alkaptonuria (AKU) is a rare inherited disease resulting from a deficiency of the enzyme homogentisate 1,2-dioxygenase which leads to the accumulation of homogentisic acid (HGA). AKU is characterized by severe cartilage degeneration, similar to that observed in osteoarthritis. Previous studies suggest that AKU is associated with alterations in cytoskeletal organization which could modulate primary cilia structure/function. This study investigated whether AKU is associated with changes in chondrocyte primary cilia and associated Hedgehog signaling which mediates cartilage degradation in osteoarthritis. Human articular chondrocytes were obtained from healthy and AKU donors. Additionally, healthy chondrocytes were treated with HGA to replicate AKU pathology (+HGA). Diseased cells exhibited shorter cilia with length reductions of 36% and 16% in AKU and +HGA chondrocytes respectively, when compared to healthy controls. Both AKU and +HGA chondrocytes demonstrated disruption of the usual cilia length regulation by actin contractility. Furthermore, the proportion of cilia with axoneme breaks and bulbous tips was increased in AKU chondrocytes consistent with defective regulation of ciliary trafficking. Distribution of the Hedgehog-related protein Arl13b along the ciliary axoneme was altered such that its localization was increased at the distal tip in AKU and +HGA chondrocytes. These changes in cilia structure/trafficking in AKU and +HGA chondrocytes were associated with a complete inability to activate Hedgehog signaling in response to exogenous ligand. Thus, we suggest that altered responsiveness to Hedgehog, as a consequence of cilia dysfunction, may be a contributing factor in the development of arthropathy highlighting the cilium as a novel target in AKU. © 2017 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc.

Human microcephaly protein RTTN interacts with STIL and is required to build full-length centrioles.

PubMed

Chen, Hsin-Yi; Wu, Chien-Ting; Tang, Chieh-Ju C; Lin, Yi-Nan; Wang, Won-Jing; Tang, Tang K

2017-08-15

Mutations in many centriolar protein-encoding genes cause primary microcephaly. Using super-resolution and electron microscopy, we find that the human microcephaly protein, RTTN, is recruited to the proximal end of the procentriole at early S phase, and is located at the inner luminal walls of centrioles. Further studies demonstrate that RTTN directly interacts with STIL and acts downstream of STIL-mediated centriole assembly. CRISPR/Cas9-mediated RTTN gene knockout in p53-deficient cells induce amplification of primitive procentriole bodies that lack the distal-half centriolar proteins, POC5 and POC1B. Additional analyses show that RTTN serves as an upstream effector of CEP295, which mediates the loading of POC1B and POC5 to the distal-half centrioles. Interestingly, the naturally occurring microcephaly-associated mutant, RTTN (A578P), shows a low affinity for STIL binding and blocks centriole assembly. These findings reveal that RTTN contributes to building full-length centrioles and illuminate the molecular mechanism through which the RTTN (A578P) mutation causes primary microcephaly.Mutations in many centriolar protein-encoding genes cause primary microcephaly. Here the authors show that human microcephaly protein RTTN directly interacts with STIL and acts downstream of STIL-mediated centriole assembly, contributing to building full-length centrioles.

Dynamic interaction of Y RNAs with chromatin and initiation proteins during human DNA replication

PubMed Central

Zhang, Alice Tianbu; Langley, Alexander R.; Christov, Christo P.; Kheir, Eyemen; Shafee, Thomas; Gardiner, Timothy J.; Krude, Torsten

2011-01-01

Non-coding Y RNAs are required for the initiation of chromosomal DNA replication in mammalian cells. It is unknown how they perform this function or if they associate with a nuclear structure during DNA replication. Here, we investigate the association of Y RNAs with chromatin and their interaction with replication proteins during DNA replication in a human cell-free system. Our results show that fluorescently labelled Y RNAs associate with unreplicated euchromatin in late G1 phase cell nuclei before the initiation of DNA replication. Following initiation, Y RNAs are displaced locally from nascent and replicated DNA present in replication foci. In intact human cells, a substantial fraction of endogenous Y RNAs are associated with G1 phase nuclei, but not with G2 phase nuclei. Y RNAs interact and colocalise with the origin recognition complex (ORC), the pre-replication complex (pre-RC) protein Cdt1, and other proteins implicated in the initiation of DNA replication. These data support a molecular ‘catch and release’ mechanism for Y RNA function during the initiation of chromosomal DNA replication, which is consistent with Y RNAs acting as replication licensing factors. PMID:21610089

Modeling of protein-anion exchange resin interaction for the human growth hormone charge variants.

PubMed

Lapelosa, Mauro; Patapoff, Thomas W; Zarraga, Isidro E

2015-12-01

Modeling ion exchange chromatography (IEC) behavior has generated significant interest because of the wide use of IEC as an analytical technique as well as a preparative protein purification process; indeed there is a need for better understanding of what drives the unique behavior of protein charge variants. We hypothesize that a complex protein molecule, which contains both hydrophobic and charged moieties, would interact strongly with an in silico designed resin through charged electrostatic patches on the surface of the protein. In the present work, variants of recombinant human growth hormone that mimic naturally-occurring deamidation products were produced and characterized in silico. The study included these four variants: rhGH, N149D, N152D, and N149D/N152D. Poisson-Boltzmann calculations were used to determine surface electrostatic potential. Metropolis Monte Carlo simulations were carried out with the resulting variants to simulate IEC systems, examining the free energy of the interaction of the protein with an in silico anion exchange column represented by polylysine polypeptide. The results show that the charge variants have different average binding energies and the free energy of interaction can be used to predict the retention time for the different variants. Copyright © 2015 Elsevier B.V. All rights reserved.

Low-intensity pulsed ultrasound stimulation promotes osteoblast differentiation through hedgehog signaling.

PubMed

Matsumoto, Kenichi; Shimo, Tsuyoshi; Kurio, Naito; Okui, Tatsuo; Ibaragi, Soichiro; Kunisada, Yuki; Obata, Kyoichi; Masui, Masanori; Pai, Pang; Horikiri, Yuu; Yamanaka, Nobuyuki; Takigawa, Masaharu; Sasaki, Akira

2018-06-01

Low-intensity pulsed ultrasound (LIPUS) has been used as an adjunct to fracture healing therapies, but the mechanisms underlying its action are not known. We reported that sonic hedgehog (SHH) signaling was activated in osteoblasts at the dynamic remodeling site of a bone fracture. Mechanical stimulation is a crucial factor in bone remodeling, and it is related to the primary cilia as a sensor of hedgehog signaling. Here we observed that LIPUS promoted callus formation in accord with Gli2-positive cells after 14 days at the mouse femur fractured site compared with a control group. An immunofluorescence analysis showed that the numbers of primary cilia and cilia/osterix double-positive osteoblasts were increased at the fracture site by LIPUS. LIPUS stimulated not only the number and the length of primary cilia, but also the levels of ciliated protein, Ift88 mRNA, and SHH, Gli1, and Gli2 in MC3T3-E1 cells. Further experiments revealed that LIPUS stimulated osteogenic differentiation in the presence of smoothened agonist (SAG) treatment. These results indicate that LIPUS stimulates osteogenic differentiation and the maturation of osteoblasts by a primary cilium-mediated activation of hedgehog signaling. © 2017 Wiley Periodicals, Inc.

Interaction of the Human Respiratory Syncytial Virus matrix protein with cellular adaptor protein complex 3 plays a critical role in trafficking.

PubMed

Ward, Casey; Maselko, Maciej; Lupfer, Christopher; Prescott, Meagan; Pastey, Manoj K

2017-01-01

Human Respiratory Syncytial Virus (HRSV) is a leading cause of bronchopneumonia in infants and the elderly. To date, knowledge of viral and host protein interactions within HRSV is limited and are critical areas of research. Here, we show that HRSV Matrix (M) protein interacts with the cellular adaptor protein complex 3 specifically via its medium subunit (AP-3Mu3A). This novel protein-protein interaction was first detected via yeast-two hybrid screen and was further confirmed in a mammalian system by immunofluorescence colocalization and co-immunoprecipitation. This novel interaction is further substantiated by the presence of a known tyrosine-based adaptor protein MU subunit sorting signal sequence, YXXФ: where Ф is a bulky hydrophobic residue, which is conserved across the related RSV M proteins. Analysis of point-mutated HRSV M derivatives indicated that AP-3Mu3A- mediated trafficking is contingent on the presence of the tyrosine residue within the YXXL sorting sequence at amino acids 197-200 of the M protein. AP-3Mu3A is up regulated at 24 hours post-infection in infected cells versus mock-infected HEp2 cells. Together, our data suggests that the AP-3 complex plays a critical role in the trafficking of HRSV proteins specifically matrix in epithelial cells. The results of this study add new insights and targets that may lead to the development of potential antivirals and attenuating mutations suitable for candidate vaccines in the future.

Arsenic inhibits hedgehog signaling during P19 cell differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Jui Tung; Bain, Lisa J., E-mail: lbain@clemson.edu; Department of Biological Sciences, Clemson University, 132 Long Hall, Clemson, SC 29634

Arsenic is a toxicant found in ground water around the world, and human exposure mainly comes from drinking water or from crops grown in areas containing arsenic in soils or water. Epidemiological studies have shown that arsenic exposure during development decreased intellectual function, reduced birth weight, and altered locomotor activity, while in vitro studies have shown that arsenite decreased muscle and neuronal cell differentiation. The sonic hedgehog (Shh) signaling pathway plays an important role during the differentiation of both neurons and skeletal muscle. The purpose of this study was to investigate whether arsenic can disrupt Shh signaling in P19 mousemore » embryonic stem cells, leading to changes muscle and neuronal cell differentiation. P19 embryonic stem cells were exposed to 0, 0.25, or 0.5 μM of sodium arsenite for up to 9 days during cell differentiation. We found that arsenite exposure significantly reduced transcript levels of genes in the Shh pathway in both a time and dose-dependent manner. This included the Shh ligand, which was decreased 2- to 3-fold, the Gli2 transcription factor, which was decreased 2- to 3-fold, and its downstream target gene Ascl1, which was decreased 5-fold. GLI2 protein levels and transcriptional activity were also reduced. However, arsenic did not alter GLI2 primary cilium accumulation or nuclear translocation. Moreover, additional extracellular SHH rescued the inhibitory effects of arsenic on cellular differentiation due to an increase in GLI binding activity. Taken together, we conclude that arsenic exposure affected Shh signaling, ultimately decreasing the expression of the Gli2 transcription factor. These results suggest a mechanism by which arsenic disrupts cell differentiation. - Highlights: • Arsenic exposure decreases sonic hedgehog pathway-related gene expression. • Arsenic decreases GLI2 protein levels and transcriptional activity in P19 cells. • Arsenic exposure does not alter the levels of SHH

Characterization of host proteins interacting with the lymphocytic choriomeningitis virus L protein.

PubMed

Khamina, Kseniya; Lercher, Alexander; Caldera, Michael; Schliehe, Christopher; Vilagos, Bojan; Sahin, Mehmet; Kosack, Lindsay; Bhattacharya, Anannya; Májek, Peter; Stukalov, Alexey; Sacco, Roberto; James, Leo C; Pinschewer, Daniel D; Bennett, Keiryn L; Menche, Jörg; Bergthaler, Andreas

2017-12-01

RNA-dependent RNA polymerases (RdRps) play a key role in the life cycle of RNA viruses and impact their immunobiology. The arenavirus lymphocytic choriomeningitis virus (LCMV) strain Clone 13 provides a benchmark model for studying chronic infection. A major genetic determinant for its ability to persist maps to a single amino acid exchange in the viral L protein, which exhibits RdRp activity, yet its functional consequences remain elusive. To unravel the L protein interactions with the host proteome, we engineered infectious L protein-tagged LCMV virions by reverse genetics. A subsequent mass-spectrometric analysis of L protein pulldowns from infected human cells revealed a comprehensive network of interacting host proteins. The obtained LCMV L protein interactome was bioinformatically integrated with known host protein interactors of RdRps from other RNA viruses, emphasizing interconnected modules of human proteins. Functional characterization of selected interactors highlighted proviral (DDX3X) as well as antiviral (NKRF, TRIM21) host factors. To corroborate these findings, we infected Trim21-/- mice with LCMV and found impaired virus control in chronic infection. These results provide insights into the complex interactions of the arenavirus LCMV and other viral RdRps with the host proteome and contribute to a better molecular understanding of how chronic viruses interact with their host.

Palmitoylation of proteins in cancer.

PubMed

Resh, Marilyn D

2017-04-15

Post-translational modification of proteins by attachment of palmitate serves as a mechanism to regulate protein localization and function in both normal and malignant cells. Given the essential role that palmitoylation plays in cancer cell signaling, approaches that target palmitoylated proteins and palmitoyl acyltransferases (PATs) have the potential for therapeutic intervention in cancer. Highlighted here are recent advances in understanding the importance of protein palmitoylation in tumorigenic pathways. A new study has uncovered palmitoylation sites within the epidermal growth factor receptor that regulate receptor trafficking, signaling and sensitivity to tyrosine kinase inhibitors. Global data analysis from nearly 150 cancer studies reveals genomic alterations in several PATs that may account for their ability to function as tumor suppressors or oncogenes. Selective inhibitors have recently been developed that target hedgehog acyltransferase (Hhat) and Porcupine (Porcn), the acyltransferases that modify hedgehog and Wnt proteins, respectively. These inhibitors, coupled with targeted knockdown of Hhat and Porcn, reveal the essential functions of fatty acylation of secreted morphogens in a wide variety of human tumors. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Potential disruption of protein-protein interactions by graphene oxide

NASA Astrophysics Data System (ADS)

Feng, Mei; Kang, Hongsuk; Yang, Zaixing; Luan, Binquan; Zhou, Ruhong

2016-06-01

Graphene oxide (GO) is a promising novel nanomaterial with a wide range of potential biomedical applications due to its many intriguing properties. However, very little research has been conducted to study its possible adverse effects on protein-protein interactions (and thus subsequent toxicity to human). Here, the potential cytotoxicity of GO is investigated at molecular level using large-scale, all-atom molecular dynamics simulations to explore the interaction mechanism between a protein dimer and a GO nanosheet oxidized at different levels. Our theoretical results reveal that GO nanosheet could intercalate between the two monomers of HIV-1 integrase dimer, disrupting the protein-protein interactions and eventually lead to dimer disassociation as graphene does [B. Luan et al., ACS Nano 9(1), 663 (2015)], albeit its insertion process is slower when compared with graphene due to the additional steric and attractive interactions. This study helps to better understand the toxicity of GO to cell functions which could shed light on how to improve its biocompatibility and biosafety for its wide potential biomedical applications.

Potential disruption of protein-protein interactions by graphene oxide.

PubMed

Feng, Mei; Kang, Hongsuk; Yang, Zaixing; Luan, Binquan; Zhou, Ruhong

2016-06-14

Graphene oxide (GO) is a promising novel nanomaterial with a wide range of potential biomedical applications due to its many intriguing properties. However, very little research has been conducted to study its possible adverse effects on protein-protein interactions (and thus subsequent toxicity to human). Here, the potential cytotoxicity of GO is investigated at molecular level using large-scale, all-atom molecular dynamics simulations to explore the interaction mechanism between a protein dimer and a GO nanosheet oxidized at different levels. Our theoretical results reveal that GO nanosheet could intercalate between the two monomers of HIV-1 integrase dimer, disrupting the protein-protein interactions and eventually lead to dimer disassociation as graphene does [B. Luan et al., ACS Nano 9(1), 663 (2015)], albeit its insertion process is slower when compared with graphene due to the additional steric and attractive interactions. This study helps to better understand the toxicity of GO to cell functions which could shed light on how to improve its biocompatibility and biosafety for its wide potential biomedical applications.

Potential disruption of protein-protein interactions by graphene oxide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feng, Mei; Kang, Hongsuk; Luan, Binquan

Graphene oxide (GO) is a promising novel nanomaterial with a wide range of potential biomedical applications due to its many intriguing properties. However, very little research has been conducted to study its possible adverse effects on protein-protein interactions (and thus subsequent toxicity to human). Here, the potential cytotoxicity of GO is investigated at molecular level using large-scale, all-atom molecular dynamics simulations to explore the interaction mechanism between a protein dimer and a GO nanosheet oxidized at different levels. Our theoretical results reveal that GO nanosheet could intercalate between the two monomers of HIV-1 integrase dimer, disrupting the protein-protein interactions andmore » eventually lead to dimer disassociation as graphene does [B. Luan et al., ACS Nano 9(1), 663 (2015)], albeit its insertion process is slower when compared with graphene due to the additional steric and attractive interactions. This study helps to better understand the toxicity of GO to cell functions which could shed light on how to improve its biocompatibility and biosafety for its wide potential biomedical applications.« less

Detection of protein complex from protein-protein interaction network using Markov clustering

NASA Astrophysics Data System (ADS)

Ochieng, P. J.; Kusuma, W. A.; Haryanto, T.

2017-05-01

Detection of complexes, or groups of functionally related proteins, is an important challenge while analysing biological networks. However, existing algorithms to identify protein complexes are insufficient when applied to dense networks of experimentally derived interaction data. Therefore, we introduced a graph clustering method based on Markov clustering algorithm to identify protein complex within highly interconnected protein-protein interaction networks. Protein-protein interaction network was first constructed to develop geometrical network, the network was then partitioned using Markov clustering to detect protein complexes. The interest of the proposed method was illustrated by its application to Human Proteins associated to type II diabetes mellitus. Flow simulation of MCL algorithm was initially performed and topological properties of the resultant network were analysed for detection of the protein complex. The results indicated the proposed method successfully detect an overall of 34 complexes with 11 complexes consisting of overlapping modules and 20 non-overlapping modules. The major complex consisted of 102 proteins and 521 interactions with cluster modularity and density of 0.745 and 0.101 respectively. The comparison analysis revealed MCL out perform AP, MCODE and SCPS algorithms with high clustering coefficient (0.751) network density and modularity index (0.630). This demonstrated MCL was the most reliable and efficient graph clustering algorithm for detection of protein complexes from PPI networks.

Quantitative study of protein-protein interactions by quartz nanopipettes

NASA Astrophysics Data System (ADS)

Tiwari, Purushottam Babu; Astudillo, Luisana; Miksovska, Jaroslava; Wang, Xuewen; Li, Wenzhi; Darici, Yesim; He, Jin

2014-08-01

In this report, protein-modified quartz nanopipettes were used to quantitatively study protein-protein interactions in attoliter sensing volumes. As shown by numerical simulations, the ionic current through the conical-shaped nanopipette is very sensitive to the surface charge variation near the pore mouth. With the appropriate modification of negatively charged human neuroglobin (hNgb) onto the inner surface of a nanopipette, we were able to detect concentration-dependent current change when the hNgb-modified nanopipette tip was exposed to positively charged cytochrome c (Cyt c) with a series of concentrations in the bath solution. Such current change is due to the adsorption of Cyt c to the inner surface of the nanopipette through specific interactions with hNgb. In contrast, a smaller current change with weak concentration dependence was observed when Cyt c was replaced with lysozyme, which does not specifically bind to hNgb. The equilibrium dissociation constant (KD) for the Cyt c-hNgb complex formation was derived and the value matched very well with the result from surface plasmon resonance measurement. This is the first quantitative study of protein-protein interactions by a conical-shaped nanopore based on charge sensing. Our results demonstrate that nanopipettes can potentially be used as a label-free analytical tool to quantitatively characterize protein-protein interactions.In this report, protein-modified quartz nanopipettes were used to quantitatively study protein-protein interactions in attoliter sensing volumes. As shown by numerical simulations, the ionic current through the conical-shaped nanopipette is very sensitive to the surface charge variation near the pore mouth. With the appropriate modification of negatively charged human neuroglobin (hNgb) onto the inner surface of a nanopipette, we were able to detect concentration-dependent current change when the hNgb-modified nanopipette tip was exposed to positively charged cytochrome c (Cyt c) with

Functional Analysis of Human Hub Proteins and Their Interactors Involved in the Intrinsic Disorder-Enriched Interactions

PubMed Central

Hu, Gang; Wu, Zhonghua

2017-01-01

Some of the intrinsically disordered proteins and protein regions are promiscuous interactors that are involved in one-to-many and many-to-one binding. Several studies have analyzed enrichment of intrinsic disorder among the promiscuous hub proteins. We extended these works by providing a detailed functional characterization of the disorder-enriched hub protein-protein interactions (PPIs), including both hubs and their interactors, and by analyzing their enrichment among disease-associated proteins. We focused on the human interactome, given its high degree of completeness and relevance to the analysis of the disease-linked proteins. We quantified and investigated numerous functional and structural characteristics of the disorder-enriched hub PPIs, including protein binding, structural stability, evolutionary conservation, several categories of functional sites, and presence of over twenty types of posttranslational modifications (PTMs). We showed that the disorder-enriched hub PPIs have a significantly enlarged number of disordered protein binding regions and long intrinsically disordered regions. They also include high numbers of targeting, catalytic, and many types of PTM sites. We empirically demonstrated that these hub PPIs are significantly enriched among 11 out of 18 considered classes of human diseases that are associated with at least 100 human proteins. Finally, we also illustrated how over a dozen specific human hubs utilize intrinsic disorder for their promiscuous PPIs. PMID:29257115

Hedgehog Signaling Promotes the Proliferation and Subsequent Hair Cell Formation of Progenitor Cells in the Neonatal Mouse Cochlea

PubMed Central

Chen, Yan; Lu, Xiaoling; Guo, Luo; Ni, Wenli; Zhang, Yanping; Zhao, Liping; Wu, Lingjie; Sun, Shan; Zhang, Shasha; Tang, Mingliang; Li, Wenyan; Chai, Renjie; Li, Huawei

2017-01-01

Hair cell (HC) loss is the major cause of permanent sensorineural hearing loss in mammals. Unlike lower vertebrates, mammalian cochlear HCs cannot regenerate spontaneously after damage, although the vestibular system does maintain limited HC regeneration capacity. Thus HC regeneration from the damaged sensory epithelium has been one of the main areas of research in the field of hearing restoration. Hedgehog signaling plays important roles during the embryonic development of the inner ear, and it is involved in progenitor cell proliferation and differentiation as well as the cell fate decision. In this study, we show that recombinant Sonic Hedgehog (Shh) protein effectively promotes sphere formation, proliferation, and differentiation of Lgr5+ progenitor cells isolated from the neonatal mouse cochlea. To further explore this, we determined the effect of Hedgehog signaling on cell proliferation and HC regeneration in cultured cochlear explant from transgenic R26-SmoM2 mice that constitutively activate Hedgehog signaling in the supporting cells of the cochlea. Without neomycin treatment, up-regulation of Hedgehog signaling did not significantly promote cell proliferation or new HC formation. However, after injury to the sensory epithelium by neomycin treatment, the over-activation of Hedgehog signaling led to significant supporting cell proliferation and HC regeneration in the cochlear epithelium explants. RNA sequencing and real-time PCR were used to compare the transcripts of the cochleae from control mice and R26-SmoM2 mice, and multiple genes involved in the proliferation and differentiation processes were identified. This study has important implications for the treatment of sensorineural hearing loss by manipulating the Hedgehog signaling pathway. PMID:29311816

Specificity of molecular interactions in transient protein-protein interaction interfaces.

PubMed

Cho, Kyu-il; Lee, KiYoung; Lee, Kwang H; Kim, Dongsup; Lee, Doheon

2006-11-15

In this study, we investigate what types of interactions are specific to their biological function, and what types of interactions are persistent regardless of their functional category in transient protein-protein heterocomplexes. This is the first approach to analyze protein-protein interfaces systematically at the molecular interaction level in the context of protein functions. We perform systematic analysis at the molecular interaction level using classification and feature subset selection technique prevalent in the field of pattern recognition. To represent the physicochemical properties of protein-protein interfaces, we design 18 molecular interaction types using canonical and noncanonical interactions. Then, we construct input vector using the frequency of each interaction type in protein-protein interface. We analyze the 131 interfaces of transient protein-protein heterocomplexes in PDB: 33 protease-inhibitors, 52 antibody-antigens, 46 signaling proteins including 4 cyclin dependent kinase and 26 G-protein. Using kNN classification and feature subset selection technique, we show that there are specific interaction types based on their functional category, and such interaction types are conserved through the common binding mechanism, rather than through the sequence or structure conservation. The extracted interaction types are C(alpha)-- H...O==C interaction, cation...anion interaction, amine...amine interaction, and amine...cation interaction. With these four interaction types, we achieve the classification success rate up to 83.2% with leave-one-out cross-validation at k = 15. Of these four interaction types, C(alpha)--H...O==C shows binding specificity for protease-inhibitor complexes, while cation-anion interaction is predominant in signaling complexes. The amine ... amine and amine...cation interaction give a minor contribution to the classification accuracy. When combined with these two interactions, they increase the accuracy by 3.8%. In the case of

The HUPO PSI's molecular interaction format--a community standard for the representation of protein interaction data.

PubMed

Hermjakob, Henning; Montecchi-Palazzi, Luisa; Bader, Gary; Wojcik, Jérôme; Salwinski, Lukasz; Ceol, Arnaud; Moore, Susan; Orchard, Sandra; Sarkans, Ugis; von Mering, Christian; Roechert, Bernd; Poux, Sylvain; Jung, Eva; Mersch, Henning; Kersey, Paul; Lappe, Michael; Li, Yixue; Zeng, Rong; Rana, Debashis; Nikolski, Macha; Husi, Holger; Brun, Christine; Shanker, K; Grant, Seth G N; Sander, Chris; Bork, Peer; Zhu, Weimin; Pandey, Akhilesh; Brazma, Alvis; Jacq, Bernard; Vidal, Marc; Sherman, David; Legrain, Pierre; Cesareni, Gianni; Xenarios, Ioannis; Eisenberg, David; Steipe, Boris; Hogue, Chris; Apweiler, Rolf

2004-02-01

A major goal of proteomics is the complete description of the protein interaction network underlying cell physiology. A large number of small scale and, more recently, large-scale experiments have contributed to expanding our understanding of the nature of the interaction network. However, the necessary data integration across experiments is currently hampered by the fragmentation of publicly available protein interaction data, which exists in different formats in databases, on authors' websites or sometimes only in print publications. Here, we propose a community standard data model for the representation and exchange of protein interaction data. This data model has been jointly developed by members of the Proteomics Standards Initiative (PSI), a work group of the Human Proteome Organization (HUPO), and is supported by major protein interaction data providers, in particular the Biomolecular Interaction Network Database (BIND), Cellzome (Heidelberg, Germany), the Database of Interacting Proteins (DIP), Dana Farber Cancer Institute (Boston, MA, USA), the Human Protein Reference Database (HPRD), Hybrigenics (Paris, France), the European Bioinformatics Institute's (EMBL-EBI, Hinxton, UK) IntAct, the Molecular Interactions (MINT, Rome, Italy) database, the Protein-Protein Interaction Database (PPID, Edinburgh, UK) and the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING, EMBL, Heidelberg, Germany).

TRAIL, Wnt, Sonic Hedgehog, TGFβ, and miRNA Signalings Are Potential Targets for Oral Cancer Therapy

PubMed Central

Farooqi, Ammad Ahmad; Shu, Chih-Wen; Huang, Hurng-Wern; Wang, Hui-Ru; Chang, Yung-Ting; Fayyaz, Sundas; Yuan, Shyng-Shiou F.; Tang, Jen-Yang

2017-01-01

Clinical studies and cancer cell models emphasize the importance of targeting therapies for oral cancer. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is highly expressed in cancer, and is a selective killing ligand for oral cancer. Signaling proteins in the wingless-type mouse mammary tumor virus (MMTV) integration site family (Wnt), Sonic hedgehog (SHH), and transforming growth factor β (TGFβ) pathways may regulate cell proliferation, migration, and apoptosis. Accordingly, the genes encoding these signaling proteins are potential targets for oral cancer therapy. In this review, we focus on recent advances in targeting therapies for oral cancer and discuss the gene targets within TRAIL, Wnt, SHH, and TGFβ signaling for oral cancer therapies. Oncogenic microRNAs (miRNAs) and tumor suppressor miRNAs targeting the genes encoding these signaling proteins are summarized, and the interactions between Wnt, SHH, TGFβ, and miRNAs are interpreted. With suitable combination treatments, synergistic effects are expected to improve targeting therapies for oral cancer. PMID:28708091

TRAIL, Wnt, Sonic Hedgehog, TGFβ, and miRNA Signalings Are Potential Targets for Oral Cancer Therapy.

PubMed

Farooqi, Ammad Ahmad; Shu, Chih-Wen; Huang, Hurng-Wern; Wang, Hui-Ru; Chang, Yung-Ting; Fayyaz, Sundas; Yuan, Shyng-Shiou F; Tang, Jen-Yang; Chang, Hsueh-Wei

2017-07-14

Clinical studies and cancer cell models emphasize the importance of targeting therapies for oral cancer. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is highly expressed in cancer, and is a selective killing ligand for oral cancer. Signaling proteins in the wingless-type mouse mammary tumor virus (MMTV) integration site family (Wnt), Sonic hedgehog (SHH), and transforming growth factor β (TGFβ) pathways may regulate cell proliferation, migration, and apoptosis. Accordingly, the genes encoding these signaling proteins are potential targets for oral cancer therapy. In this review, we focus on recent advances in targeting therapies for oral cancer and discuss the gene targets within TRAIL, Wnt, SHH, and TGFβ signaling for oral cancer therapies. Oncogenic microRNAs (miRNAs) and tumor suppressor miRNAs targeting the genes encoding these signaling proteins are summarized, and the interactions between Wnt, SHH, TGFβ, and miRNAs are interpreted. With suitable combination treatments, synergistic effects are expected to improve targeting therapies for oral cancer.

Corynebacterial pneumonia in an African hedgehog.

PubMed

Raymond, J T; Williams, C; Wu, C C

1998-04-01

A 3-mo-old, male African hedgehog (Atelerix albiventris) was anorectic and lethargic for a period of 3 days prior to death. Necropys revealed lungs that were diffusely firm, dark red, and dorsally adhered by fibrinous tags to the pericardial sac. Histopathology revealed necrosuppurative bronchopneumonia with pulmonary abscesses and suppurative pericarditis and myocarditis. A Corynebacterium sp. was isolated from the lungs. We believe this is the first reported case of corynebacterial pneumonia in an African hedgehog.

Chylous ascites in a hedgehog (Atelerix albiventris).

PubMed

Roh, Yoon-Seok; Kim, Eun-Ju; Cho, Ara; Kim, Min-Su; Cho, Ho-Seong; Lim, Chae Woong; Kim, Bumseok

2014-12-01

An African pygmy hedgehog (Atelerix albiventris) was diagnosed as chylous ascites with biliary cirrhosis. Abdomenocentesis revealed a milky fluid with a 324 mg/dl triglyceride level. On serum biochemical examination, the hedgehog had hypoalbuminemia, hypoglycemia, and high blood urea nitrogen. There was no cytologic or genomic evidence of infection, and a blood culture was negative. Histopathologic examination revealed a liver with proliferative bile ducts that were often surrounded by prominent septa of fibrous connective tissue. In the area of ductular reaction, proliferative cells positive for CD66, an embryogenic antigen of epithelial cells, were revealed. The potential association between chylous ascites and liver cirrhosis is undetermined but could be an aspect of future study. This is the first description of chylous ascites in a hedgehog.

Adult neurogenesis in the hedgehog (Erinaceus concolor) and mole (Talpa europaea).

PubMed

Bartkowska, K; Turlejski, K; Grabiec, M; Ghazaryan, A; Yavruoyan, E; Djavadian, R L

2010-01-01

We investigated adult neurogenesis in two species of mammals belonging to the superorder Laurasiatheria, the southern white-breasted hedgehog (order Erinaceomorpha, species Erinaceus concolor) from Armenia and the European mole (order Soricomorpha, species Talpa europaea) from Poland. Neurogenesis in the brain of these species was examined immunohistochemically, using the endogenous markers doublecortin (DCX) and Ki-67, which are highly conserved among species. We found that in both the hedgehog and mole, like in the majority of earlier investigated mammals, neurogenesis continues in the subventricular zone (SVZ) of the lateral ventricles and in the dentate gyrus (DG). In the DG of both species, DCX-expressing cells and Ki-67-labeled cells were present in the subgranular and granular layers. In the mole, a strong bundle of DCX-labeled processes, presumably axons of granule cells, was observed in the center of the hilus. Proliferating cells (expressing Ki-67) were identified in the SVZ of lateral ventricles of both species, but neuronal precursor cells (expressing DCX) were also observed in the olfactory bulb (OB). In both species, the vast majority of cells expressing DCX in the OB were granule cells with radially orientated dendrites, although some periglomerular cells surrounding the glomeruli were also labeled. In addition, this paper is the first to show DCX-labeled fibers in the anterior commissure of the hedgehog and mole. These fibers must be axons of new neurons making interhemispheric connections between the two OB or piriform (olfactory) cortices. DCX-expressing neurons were observed in the striatum and piriform cortex of both hedgehog and mole. We postulate that in both species a fraction of cells newly generated in the SVZ migrates along the rostral migratory stream to the piriform cortex. This pattern of migration resembles that of the 'second-wave neurons' generated during embryonal development of the neocortex rather than the pattern observed during

Palmitoylation is required for the production of a soluble multimeric Hedgehog protein complex and long-range signaling in vertebrates

PubMed Central

Chen, Miao-Hsueh; Li, Ya-Jun; Kawakami, Takatoshi; Xu, Shan-Mei; Chuang, Pao-Tien

2004-01-01

Hedgehog (Hh) signaling plays a major role in multiple aspects of embryonic development. A key issue in Hh signaling is to elucidate the molecular mechanism by which a Hh protein morphogen gradient is formed despite its membrane association. In this study, we used a combination of genetic, cellular, and biochemical approaches to address the role of lipid modifications in long-range vertebrate Hh signaling. Our molecular analysis of knockout mice deficient in Skn, the murine homolog of the Drosophila ski gene, which catalyzes Hh palmitoylation, and gene-targeted mice producing a nonpalmitoylated form of Shh indicates that Hh palmitoylation is essential for its activity as well as the generation of a protein gradient in the developing embryos. Furthermore, our biochemical data show that Hh lipid modifications are required for producing a soluble multimeric protein complex, which constitutes the major active component for Hh signaling. These results suggest that soluble Hh multimeric complex travels in the morphogenetic field to activate Hh signaling in distant Hh-responsive cells. PMID:15075292

Evidence for the interaction of the regulatory protein Ki-1/57 with p53 and its interacting proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nery, Flavia C.; Departamento de Genetica e Evolucao, Universidade Estadual de Campinas, Campinas, SP; Rui, Edmilson

Ki-1/57 is a cytoplasmic and nuclear phospho-protein of 57 kDa and interacts with the adaptor protein RACK1, the transcription factor MEF2C, and the chromatin remodeling factor CHD3, suggesting that it might be involved in the regulation of transcription. Here, we describe yeast two-hybrid studies that identified a total of 11 proteins interacting with Ki-1/57, all of which interact or are functionally associated with p53 or other members of the p53 family of proteins. We further found that Ki-1/57 is able to interact with p53 itself in the yeast two-hybrid system when the interaction was tested directly. This interaction could bemore » confirmed by pull down assays with purified proteins in vitro and by reciprocal co-immunoprecipitation assays from the human Hodgkin analogous lymphoma cell line L540. Furthermore, we found that the phosphorylation of p53 by PKC abolishes its interaction with Ki-1/57 in vitro.« less

Protein interaction networks from literature mining

NASA Astrophysics Data System (ADS)

Ihara, Sigeo

2005-03-01

The ability to accurately predict and understand physiological changes in the biological network system in response to disease or drug therapeutics is of crucial importance in life science. The extensive amount of gene expression data generated from even a single microarray experiment often proves difficult to fully interpret and comprehend the biological significance. An increasing knowledge of protein interactions stored in the PubMed database, as well as the advancement of natural language processing, however, makes it possible to construct protein interaction networks from the gene expression information that are essential for understanding the biological meaning. From the in house literature mining system we have developed, the protein interaction network for humans was constructed. By analysis based on the graph-theoretical characterization of the total interaction network in literature, we found that the network is scale-free and semantic long-ranged interactions (i.e. inhibit, induce) between proteins dominate in the total interaction network, reducing the degree exponent. Interaction networks generated based on scientific text in which the interaction event is ambiguously described result in disconnected networks. In contrast interaction networks based on text in which the interaction events are clearly stated result in strongly connected networks. The results of protein-protein interaction networks obtained in real applications from microarray experiments are discussed: For example, comparisons of the gene expression data indicative of either a good or a poor prognosis for acute lymphoblastic leukemia with MLL rearrangements, using our system, showed newly discovered signaling cross-talk.

Proteomic identification of dysferlin-interacting protein complexes in human vascular endothelium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leung, Cleo; Utokaparch, Soraya; Sharma, Arpeeta

2011-11-18

Highlights: Black-Right-Pointing-Pointer Bi-directional (inward and outward) movement of GFP-dysferlin in COS-7 cells. Black-Right-Pointing-Pointer Dysferlin interacts with key signaling proteins for transcytosis in EC. Black-Right-Pointing-Pointer Dysferlin mediates trafficking of vesicles carrying protein cargos in EC. -- Abstract: Dysferlin is a membrane-anchored protein known to facilitate membrane repair in skeletal muscles following mechanical injury. Mutations of dysferlin gene impair sarcolemma integrity, a hallmark of certain forms of muscular dystrophy in patients. Dysferlin contains seven calcium-dependent C2 binding domains, which are required to promote fusion of intracellular membrane vesicles. Emerging evidence reveal the unexpected expression of dysferlin in non-muscle, non-mechanically active tissues, suchmore » as endothelial cells, which cast doubts over the belief that ferlin proteins act exclusively as membrane repair proteins. We and others have shown that deficient trafficking of membrane bound proteins in dysferlin-deficient cells, suggesting that dysferlin might mediate trafficking of client proteins. Herein, we describe the intracellular trafficking and movement of GFP-dysferlin positive vesicles in unfixed reconstituted cells using live microscopy. By performing GST pull-down assays followed by mass spectrometry, we identified dysferlin binding protein complexes in human vascular endothelial cells. Together, our data further support the claims that dysferlin not only mediates membrane repair but also trafficking of client proteins, ultimately, help bridging dysferlinopathies to aberrant membrane signaling.« less

Suppressor of Fused Chaperones Gli Proteins To Generate Transcriptional Responses to Sonic Hedgehog Signaling

PubMed Central

Zhang, Ziyu; Shen, Longyan; Law, Kelvin; Zhang, Zengdi; Liu, Xiaotong; Hua, Hu; Li, Sanen; Huang, Huijie; Yue, Shen; Hui, Chi-chung

2016-01-01

ABSTRACT Cellular responses to the graded Sonic Hedgehog (Shh) morphogenic signal are orchestrated by three Gli genes that give rise to both transcription activators and repressors. An essential downstream regulator of the pathway, encoded by the tumor suppressor gene Suppressor of fused (Sufu), plays critical roles in the production, trafficking, and function of Gli proteins, but the mechanism remains controversial. Here, we show that Sufu is upregulated in active Shh responding tissues and accompanies Gli activators translocating into and Gli repressors out of the nucleus. Trafficking of Sufu to the primary cilium, potentiated by Gli activators but not repressors, was found to be coupled to its nuclear import. We have identified a nuclear export signal (NES) motif of Sufu in juxtaposition to the protein kinase A (PKA) and glycogen synthase kinase 3 (GSK3) dual phosphorylation sites and show that Sufu binds the chromatin with both Gli1 and Gli3. Close comparison of neural tube development among individual Ptch1−/−, Sufu−/−, and Ptch1−/−; Sufu−/− double mutant embryos indicates that Sufu is critical for the maximal activation of Shh signaling essential to the specification of the most-ventral neurons. These data define Sufu as a novel class of molecular chaperone required for every aspect of Gli regulation and function. PMID:27849569

Human testicular protein TPX1/CRISP-2: localization in spermatozoa, fate after capacitation and relevance for gamete interaction.

PubMed

Busso, D; Cohen, D J; Hayashi, M; Kasahara, M; Cuasnicú, P S

2005-04-01

Testicular protein Tpx-1, also known as CRISP-2, is a cysteine-rich secretory protein specifically expressed in the male reproductive tract. Since the information available on the human protein is limited to the identification and expression of its gene, in this work we have studied the presence and localization of human Tpx-1 (TPX1) in sperm, its fate after capacitation and acrosome reaction (AR), and its possible involvement in gamete interaction. Indirect immunofluorescence studies revealed the absence of significant staining in live or fixed non-permeabilized sperm, in contrast to a clear labelling in the acrosomal region of permeabilized sperm. These results, together with complementary evidence from protein extraction procedures strongly support that TPX1 would be mainly an intra-acrosomal protein in fresh sperm. After in vitro capacitation and ionophore-induced AR, TPX1 remained associated with the equatorial segment of the acrosome. The lack of differences in the electrophoretic mobility of TPX1 before and after capacitation and AR indicates that the protein would not undergo proteolytical modifications during these processes. The possible involvement of TPX1 in gamete interaction was evaluated by the hamster oocyte penetration test. The presence of anti-TPX1 during gamete co-incubation produced a significant and dose-dependent inhibition in the percentage of penetrated zona-free hamster oocytes without affecting sperm motility, the AR or sperm binding to the oolema. Together, these results indicate that human TPX1 would be a component of the sperm acrosome that remains associated with sperm after capacitation and AR, and is relevant for sperm-oocyte interaction.

Intestinal cell kinase, a protein associated with endocrine-cerebro-osteodysplasia syndrome, is a key regulator of cilia length and Hedgehog signaling.

PubMed

Moon, Heejung; Song, Jieun; Shin, Jeong-Oh; Lee, Hankyu; Kim, Hong-Kyung; Eggenschwiller, Jonathan T; Bok, Jinwoong; Ko, Hyuk Wan

2014-06-10

Endocrine-cerebro-osteodysplasia (ECO) syndrome is a recessive genetic disorder associated with multiple congenital defects in endocrine, cerebral, and skeletal systems that is caused by a missense mutation in the mitogen-activated protein kinase-like intestinal cell kinase (ICK) gene. In algae and invertebrates, ICK homologs are involved in flagellar formation and ciliogenesis, respectively. However, it is not clear whether this role of ICK is conserved in mammals and how a lack of functional ICK results in the characteristic phenotypes of human ECO syndrome. Here, we generated Ick knockout mice to elucidate the precise role of ICK in mammalian development and to examine the pathological mechanisms of ECO syndrome. Ick null mouse embryos displayed cleft palate, hydrocephalus, polydactyly, and delayed skeletal development, closely resembling ECO syndrome phenotypes. In cultured cells, down-regulation of Ick or overexpression of kinase-dead or ECO syndrome mutant ICK resulted in an elongation of primary cilia and abnormal Sonic hedgehog (Shh) signaling. Wild-type ICK proteins were generally localized in the proximal region of cilia near the basal bodies, whereas kinase-dead ICK mutant proteins accumulated in the distal part of bulged ciliary tips. Consistent with these observations in cultured cells, Ick knockout mouse embryos displayed elongated cilia and reduced Shh signaling during limb digit patterning. Taken together, these results indicate that ICK plays a crucial role in controlling ciliary length and that ciliary defects caused by a lack of functional ICK leads to abnormal Shh signaling, resulting in congenital disorders such as ECO syndrome.

BioPlex Display: An Interactive Suite for Large-Scale AP-MS Protein-Protein Interaction Data.

PubMed

Schweppe, Devin K; Huttlin, Edward L; Harper, J Wade; Gygi, Steven P

2018-01-05

The development of large-scale data sets requires a new means to display and disseminate research studies to large audiences. Knowledge of protein-protein interaction (PPI) networks has become a principle interest of many groups within the field of proteomics. At the confluence of technologies, such as cross-linking mass spectrometry, yeast two-hybrid, protein cofractionation, and affinity purification mass spectrometry (AP-MS), detection of PPIs can uncover novel biological inferences at a high-throughput. Thus new platforms to provide community access to large data sets are necessary. To this end, we have developed a web application that enables exploration and dissemination of the growing BioPlex interaction network. BioPlex is a large-scale interactome data set based on AP-MS of baits from the human ORFeome. The latest BioPlex data set release (BioPlex 2.0) contains 56 553 interactions from 5891 AP-MS experiments. To improve community access to this vast compendium of interactions, we developed BioPlex Display, which integrates individual protein querying, access to empirical data, and on-the-fly annotation of networks within an easy-to-use and mobile web application. BioPlex Display enables rapid acquisition of data from BioPlex and development of hypotheses based on protein interactions.

Evolutionarily conserved heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins functionally interact with human and Drosophila TAR DNA-binding protein 43 (TDP-43).

PubMed

Romano, Maurizio; Buratti, Emanuele; Romano, Giulia; Klima, Raffaella; Del Bel Belluz, Lisa; Stuani, Cristiana; Baralle, Francisco; Feiguin, Fabian

2014-03-07

Human TDP-43 represents the main component of neuronal inclusions found in patients with neurodegenerative diseases, especially frontotemporal lobar degeneration and amyotrophic lateral sclerosis. In vitro and in vivo studies have shown that the TAR DNA-binding protein 43 (TDP-43) Drosophila ortholog (TBPH) can biochemically and functionally overlap the properties of the human factor. The recent direct implication of the human heterogeneous nuclear ribonucleoproteins (hnRNPs) A2B1 and A1, known TDP-43 partners, in the pathogenesis of multisystem proteinopathy and amyotrophic lateral sclerosis supports the hypothesis that the physical and functional interplay between TDP-43 and hnRNP A/B orthologs might play a crucial role in the pathogenesis of neurodegenerative diseases. To test this hypothesis and further validate the fly system as a useful model to study this type of diseases, we have now characterized human TDP-43 and Drosophila TBPH similarity in terms of protein-protein interaction pathways. In this work we show that TDP-43 and TBPH share the ability to associate in vitro with Hrp38/Hrb98DE/CG9983, the fruit fly ortholog of the human hnRNP A1/A2 factors. Interestingly, the protein regions of TDP-43 and Hrp38 responsible for reciprocal interactions are conserved through evolution. Functionally, experiments in HeLa cells demonstrate that TDP-43 is necessary for the inhibitory activity of Hrp38 on splicing. Finally, Drosophila in vivo studies show that Hrp38 deficiency produces locomotive defects and life span shortening in TDP-43 with and without animals. These results suggest that hnRNP protein levels can play a modulatory role on TDP-43 functions.

Profiling lethal factor interacting proteins from human stomach using T7 phage display screening.

PubMed

Cardona-Correa, Albin; Rios-Velazquez, Carlos

2016-05-01

The anthrax lethal factor (LF) is a zinc dependent metalloproteinase that cleaves the majority of mitogen-activated protein kinase kinases and a member of NOD-like receptor proteins, inducing cell apoptosis. Despite efforts to fully understand the Bacillus anthracis toxin components, the gastrointestinal (GI) anthrax mechanisms have not been fully elucidated. Previous studies demonstrated gastric ulceration, and a substantial bacterial growth rate in Peyer's patches. However, the complete molecular pathways of the disease that results in tissue damage by LF proteolytic activity remains unclear. In the present study, to identify the profile of the proteins potentially involved in GI anthrax, protein‑protein interactions were investigated using human stomach T7 phage display (T7PD) cDNA libraries. T7PD is a high throughput technique that allows the expression of cloned DNA sequences as peptides on the phage surface, enabling the selection and identification of protein ligands. A wild type and mutant LF (E687A) were used to differentiate interaction sites. A total of 124 clones were identified from 194 interacting‑phages, at both the DNA and protein level, by in silico analysis. Databases revealed that the selected candidates were proteins from different families including lipase, peptidase‑A1 and cation transport families, among others. Furthermore, individual T7PD candidates were tested against LF in order to detect their specificity to the target molecule, resulting in 10 LF‑interacting peptides. With a minimum concentration of LF for interaction at 1 µg/ml, the T7PD isolated pepsin A3 pre‑protein (PAP) demonstrated affinity to both types of LF. In addition, PAP was isolated in various lengths for the same protein, exhibiting common regions following PRALINE alignment. These findings will help elucidate and improve the understanding of the molecular pathogenesis of GI anthrax, and aid in the development of potential therapeutic agents.

Topological and organizational properties of the products of house-keeping and tissue-specific genes in protein-protein interaction networks.

PubMed

Lin, Wen-Hsien; Liu, Wei-Chung; Hwang, Ming-Jing

2009-03-11

Human cells of various tissue types differ greatly in morphology despite having the same set of genetic information. Some genes are expressed in all cell types to perform house-keeping functions, while some are selectively expressed to perform tissue-specific functions. In this study, we wished to elucidate how proteins encoded by human house-keeping genes and tissue-specific genes are organized in human protein-protein interaction networks. We constructed protein-protein interaction networks for different tissue types using two gene expression datasets and one protein-protein interaction database. We then calculated three network indices of topological importance, the degree, closeness, and betweenness centralities, to measure the network position of proteins encoded by house-keeping and tissue-specific genes, and quantified their local connectivity structure. Compared to a random selection of proteins, house-keeping gene-encoded proteins tended to have a greater number of directly interacting neighbors and occupy network positions in several shortest paths of interaction between protein pairs, whereas tissue-specific gene-encoded proteins did not. In addition, house-keeping gene-encoded proteins tended to connect with other house-keeping gene-encoded proteins in all tissue types, whereas tissue-specific gene-encoded proteins also tended to connect with other tissue-specific gene-encoded proteins, but only in approximately half of the tissue types examined. Our analysis showed that house-keeping gene-encoded proteins tend to occupy important network positions, while those encoded by tissue-specific genes do not. The biological implications of our findings were discussed and we proposed a hypothesis regarding how cells organize their protein tools in protein-protein interaction networks. Our results led us to speculate that house-keeping gene-encoded proteins might form a core in human protein-protein interaction networks, while clusters of tissue-specific gene

SPOP suppresses tumorigenesis by regulating Hedgehog/Gli2 signaling pathway in gastric cancer.

PubMed

Zeng, Chunyan; Wang, Yao; Lu, Quqin; Chen, Jiang; Zhang, Junyan; Liu, Tao; Lv, Nonghua; Luo, Shiwen

2014-09-11

Recent evidence suggests that aberrant activation of Hedgehog (Hh) signaling by Gli transcription factors is characteristic of a variety of aggressive human carcinomas including gastric cancer. Speckle-type POZ protein, SPOP, is an E3 ubiquitin ligase adaptor, and it is found to inhibit oncogenic signaling. However, the molecular mechanisms are largely unknown. In this study, we characterized the expression of SPOP in 88 pairs of gastric cancer tissues and adjacent tissues by immunohistochemical staining and Western blotting. The relationship between SPOP expression and clinical pathologic factors was analyzed. Transfected gastric cancer cell lines were used in cell viability, wound healing and colony formation assays. The interaction of SPOP with Gli2 and other related apoptotic proteins was assessed by immunoprecipitation, Western blotting, real-time PCR and dual luciferase reporter assays. Intracellular interaction of SPOP and Gli2 was visualized by immunofluorescent staining in gastric cancer cells. Immunohistochemical staining of SPOP can be detected in gastric cancer tissues but much less than adjacent gastric tissues (P < 0.01). High SPOP expression is negatively correlated with lymph node metastasis, poor histological differentiation, and tumor malignancy according to TNM staging. In vitro experiments revealed that over-expression of SPOP prevented tumor cells from proliferation, migration and colony formation in gastric cancer cell lines. Likewise, repression of SPOP promoted cell viability, migration, proliferation, and attenuated apoptosis. Mechanistic studies revealed that increasing SPOP accelerated Gli2 degradation but regardless of Gli2 synthesis. Furthermore, cytoplasmic Gli2 decreased markedly along with the abundant expression of SPOP in MKN45 cells. Our findings indicate that SPOP plays critical roles in suppressing gastric tumorigenesis through inhibiting Hh/Gli2 signaling pathway. It may provide an alternative strategy for developing

A Screen for Modifiers of Hedgehog Signaling in Drosophila melanogaster Identifies swm and mts

PubMed Central

Casso, David J.; Liu, Songmei; Iwaki, D. David; Ogden, Stacey K.; Kornberg, Thomas B.

2008-01-01

Signaling by Hedgehog (Hh) proteins shapes most tissues and organs in both vertebrates and invertebrates, and its misregulation has been implicated in many human diseases. Although components of the signaling pathway have been identified, key aspects of the signaling mechanism and downstream targets remain to be elucidated. We performed an enhancer/suppressor screen in Drosophila to identify novel components of the pathway and identified 26 autosomal regions that modify a phenotypic readout of Hh signaling. Three of the regions include genes that contribute constituents to the pathway—patched, engrailed, and hh. One of the other regions includes the gene microtubule star (mts) that encodes a subunit of protein phosphatase 2A. We show that mts is necessary for full activation of Hh signaling. A second region includes the gene second mitotic wave missing (swm). swm is recessive lethal and is predicted to encode an evolutionarily conserved protein with RNA binding and Zn+ finger domains. Characterization of newly isolated alleles indicates that swm is a negative regulator of Hh signaling and is essential for cell polarity. PMID:18245841

Computational Analysis of Host-Pathogen Protein Interactions between Humans and Different Strains of Enterohemorrhagic Escherichia coli.

PubMed

Bose, Tungadri; Venkatesh, K V; Mande, Sharmila S

2017-01-01

Serotype O157:H7, an enterohemorrhagic Escherichia coli (EHEC), is known to cause gastrointestinal and systemic illnesses ranging from diarrhea and hemorrhagic colitis to potentially fatal hemolytic uremic syndrome. Specific genetic factors like ompA, nsrR , and LEE genes are known to play roles in EHEC pathogenesis. However, these factors are not specific to EHEC and their presence in several non-pathogenic strains indicates that additional factors are involved in pathogenicity. We propose a comprehensive effort to screen for such potential genetic elements, through investigation of biomolecular interactions between E. coli and their host. In this work, an in silico investigation of the protein-protein interactions (PPIs) between human cells and four EHEC strains (viz., EDL933, Sakai, EC4115, and TW14359) was performed in order to understand the virulence and host-colonization strategies of these strains. Potential host-pathogen interactions (HPIs) between human cells and the "non-pathogenic" E. coli strain MG1655 were also probed to evaluate whether and how the variations in the genomes could translate into altered virulence and host-colonization capabilities of the studied bacterial strains. Results indicate that a small subset of HPIs are unique to the studied pathogens and can be implicated in virulence. This subset of interactions involved E. coli proteins like YhdW, ChuT, EivG, and HlyA. These proteins have previously been reported to be involved in bacterial virulence. In addition, clear differences in lineage and clade-specific HPI profiles could be identified. Furthermore, available gene expression profiles of the HPI-proteins were utilized to estimate the proportion of proteins which may be involved in interactions. We hypothesized that a cumulative score of the ratios of bound:unbound proteins (involved in HPIs) would indicate the extent of colonization. Thus, we designed the Host Colonization Index (HCI) measure to determine the host colonization

Quantitative study of protein-protein interactions by quartz nanopipettes.

PubMed

Tiwari, Purushottam Babu; Astudillo, Luisana; Miksovska, Jaroslava; Wang, Xuewen; Li, Wenzhi; Darici, Yesim; He, Jin

2014-09-07

In this report, protein-modified quartz nanopipettes were used to quantitatively study protein-protein interactions in attoliter sensing volumes. As shown by numerical simulations, the ionic current through the conical-shaped nanopipette is very sensitive to the surface charge variation near the pore mouth. With the appropriate modification of negatively charged human neuroglobin (hNgb) onto the inner surface of a nanopipette, we were able to detect concentration-dependent current change when the hNgb-modified nanopipette tip was exposed to positively charged cytochrome c (Cyt c) with a series of concentrations in the bath solution. Such current change is due to the adsorption of Cyt c to the inner surface of the nanopipette through specific interactions with hNgb. In contrast, a smaller current change with weak concentration dependence was observed when Cyt c was replaced with lysozyme, which does not specifically bind to hNgb. The equilibrium dissociation constant (KD) for the Cyt c-hNgb complex formation was derived and the value matched very well with the result from surface plasmon resonance measurement. This is the first quantitative study of protein-protein interactions by a conical-shaped nanopore based on charge sensing. Our results demonstrate that nanopipettes can potentially be used as a label-free analytical tool to quantitatively characterize protein-protein interactions.

Cryptosporidium erinacei and C. parvum in a group of overwintering hedgehogs.

PubMed

Hofmannová, Lada; Hauptman, Karel; Huclová, Kristýna; Květoňová, Dana; Sak, Bohumil; Kváč, Martin

2016-10-01

This study describes cryptosporidiosis in an overwintering group of 15 European hedgehogs (Erinaceus europaeus), comprising 3 adults and 12 juveniles. Four juvenile hedgehogs were hospitalised with anorexia, malodorous diarrhoea and dehydration. Immediate parasitological examinations revealed the presence of Cryptosporidium sp. in these animals and also in 5 other juveniles. All hedgehogs were coproscopically monitored for 4 months over the winter season. Shedding of Cryptosporidium oocysts persisted from 6 to 70 days. Repeated shedding of Cryptosporidium oocysts occurred in 3 animals after 4 months subsequent to the first outbreak. Clinical signs were observed only at the beginning of the outbreak (apathy, anorexia, general weakness, mild dehydration, and malodorous faeces with changed consistence - soft/diarrhoea) in the 4 hospitalised juveniles. Overall 11 hedgehogs were Cryptosporidium-positive, both microscopically and by PCR methods. Sequence analyses of SSU rRNA and gp60 genes revealed the presence of C. parvum IIdA18G1 subtype in all positive hedgehogs. Moreover, 3 hedgehogs had a mixed infection of the zoonotic C. parvum and C. erinacei XIIIaA19R13 subtype. Cryptosporidium infections can be rapidly spread among debilitated animals and the positive hedgehogs released back into the wild can be a source of the infection for individuals weakened after hibernation. Copyright © 2016 Elsevier GmbH. All rights reserved.

Targeting the Hedgehog pathway in cancer: can the spines be smoothened?

PubMed

Ailles, Laurie; Siu, Lillian L

2011-04-15

Aberrant Hedgehog (Hh) pathway signaling has been suggested to play a role in the development of multiple solid tumors and hematologic malignancies. GDC-0449 is a novel first-in-human, first-in-class smoothened (SMO) inhibitor, which has completed its phase I evaluation and achieved proof of concept in tumors with Hh pathway mutations. ©2011 AACR.

WHEN AND WHY DO HEDGEHOGS AND FOXES DIFFER?

PubMed

Keil, Frank C

2010-01-01

Philip E. Tetlock's finding that "hedgehog" experts (those with one big theory) are worse predictors than "foxes" (those with multiple, less comprehensive theories) offers fertile ground for future research. Are experts as likely to exhibit hedgehog- or fox-like tendencies in areas that call for explanatory, diagnostic, and skill-based expertise-as they did when Tetlock called on experts to make predictions? Do particular domains of expertise curtail or encourage different styles of expertise? Can we trace these different styles to childhood? Finally, can we nudge hedgehogs to be more like foxes? Current research can only grope at the answers to these questions, but they are essential to gauging the health of expert political judgment.

Non-interacting surface solvation and dynamics in protein-protein interactions.

PubMed

Visscher, Koen M; Kastritis, Panagiotis L; Bonvin, Alexandre M J J

2015-03-01

Protein-protein interactions control a plethora of cellular processes, including cell proliferation, differentiation, apoptosis, and signal transduction. Understanding how and why proteins interact will inevitably lead to novel structure-based drug design methods, as well as design of de novo binders with preferred interaction properties. At a structural and molecular level, interface and rim regions are not enough to fully account for the energetics of protein-protein binding, even for simple lock-and-key rigid binders. As we have recently shown, properties of the global surface might also play a role in protein-protein interactions. Here, we report on molecular dynamics simulations performed to understand solvent effects on protein-protein surfaces. We compare properties of the interface, rim, and non-interacting surface regions for five different complexes and their free components. Interface and rim residues become, as expected, less mobile upon complexation. However, non-interacting surface appears more flexible in the complex. Fluctuations of polar residues are always lower compared with charged ones, independent of the protein state. Further, stable water molecules are often observed around polar residues, in contrast to charged ones. Our analysis reveals that (a) upon complexation, the non-interacting surface can have a direct entropic compensation for the lower interface and rim entropy and (b) the mobility of the first hydration layer, which is linked to the stability of the protein-protein complex, is influenced by the local chemical properties of the surface. These findings corroborate previous hypotheses on the role of the hydration layer in shielding protein-protein complexes from unintended protein-protein interactions. © 2014 Wiley Periodicals, Inc.

Metabolites in vertebrate Hedgehog signaling.

PubMed

Roberg-Larsen, Hanne; Strand, Martin Frank; Krauss, Stefan; Wilson, Steven Ray

2014-04-11

The Hedgehog (HH) signaling pathway is critical in embryonic development, stem cell biology, tissue homeostasis, chemoattraction and synapse formation. Irregular HH signaling is associated with a number of disease conditions including congenital disorders and cancer. In particular, deregulation of HH signaling has been linked to skin, brain, lung, colon and pancreatic cancers. Key mediators of the HH signaling pathway are the 12-pass membrane protein Patched (PTC), the 7-pass membrane protein Smoothened (SMO) and the GLI transcription factors. PTC shares homology with the RND family of small-molecule transporters and it has been proposed that it interferes with SMO through metabolites. Although a conclusive picture is lacking, substantial efforts are made to identify and understand natural metabolites/sterols, including cholesterol, vitamin D3, oxysterols and glucocorticoides, that may be affected by, or influence the HH signaling cascade at the level of PTC and SMO. In this review we will elaborate the role of metabolites in HH signaling with a focus on oxysterols, and discuss advancements in modern analytical approaches in the field. Copyright © 2014 Elsevier Inc. All rights reserved.

Dengue-2 structural proteins associate with human proteins to produce a coagulation and innate immune response biased interactome.

PubMed

Folly, Brenda B; Weffort-Santos, Almeriane M; Fathman, C G; Soares, Luis R B

2011-01-31

Dengue virus infection is a public health threat to hundreds of millions of individuals in the tropical regions of the globe. Although Dengue infection usually manifests itself in its mildest, though often debilitating clinical form, dengue fever, life-threatening complications commonly arise in the form of hemorrhagic shock and encephalitis. The etiological basis for the virus-induced pathology in general, and the different clinical manifestations in particular, are not well understood. We reasoned that a detailed knowledge of the global biological processes affected by virus entry into a cell might help shed new light on this long-standing problem. A bacterial two-hybrid screen using DENV2 structural proteins as bait was performed, and the results were used to feed a manually curated, global dengue-human protein interaction network. Gene ontology and pathway enrichment, along with network topology and microarray meta-analysis, were used to generate hypothesis regarding dengue disease biology. Combining bioinformatic tools with two-hybrid technology, we screened human cDNA libraries to catalogue proteins physically interacting with the DENV2 virus structural proteins, Env, cap and PrM. We identified 31 interacting human proteins representing distinct biological processes that are closely related to the major clinical diagnostic feature of dengue infection: haemostatic imbalance. In addition, we found dengue-binding human proteins involved with additional key aspects, previously described as fundamental for virus entry into cells and the innate immune response to infection. Construction of a DENV2-human global protein interaction network revealed interesting biological properties suggested by simple network topology analysis. Our experimental strategy revealed that dengue structural proteins interact with human protein targets involved in the maintenance of blood coagulation and innate anti-viral response processes, and predicts that the interaction of dengue

Ectoparasitic infestations of the European hedgehog (Erinaceus europaeus) in Urmia city, Iran: First report

PubMed Central

Gorgani-Firouzjaee, Tahmineh; Pour-Reza, Behzad; Naem, Soraya; Tavassoli, Mousa

2013-01-01

Hedgehogs are small, nocturnal mammals that become popular in the world and have significant role in transmission of zoonotic agents. Some of the agents are transmitted by ticks and fleas such as rickettsial agents. For these reason, a survey on ectoparasites in European hedgehog (Erinaceus europaeus) carried out between April 2006 and December 2007 from different parts of Urmia city, west Azerbaijan, Iran. After being euthanized external surface of body of animals was precisely considered for ectoparasites, and arthropods were collected and stored in 70% ethanol solution. Out of 34 hedgehogs 23 hedgehogs (67.70%) were infested with ticks (Rhipicephalus turanicus). Fleas of the species Archaeopsylla erinacei were found on 19 hedgehogs of 34 hedgehogs (55.90%). There was no significant differences between sex of ticks (p > 0.05) but found in fleas (p < 0.05). The prevalence of infestation in sexes and the body condition of hedgehogs (small, medium and large) with ticks and fleas did not show significant differences (p > 0.05). Highest occurrence of infestation in both tick and flea was in June. Among three seasons of hedgehog collection significant differences was observed (p < 0.05). The result of our survey revealed that infestation rate in hedgehog was high. According to zoonotic importance of this ectoparasite and ability to transmission of some pathogens, more studies are needed to investigate hedgehog parasites in different parts of Iran. PMID:25653796

Protein annotation from protein interaction networks and Gene Ontology.

PubMed

Nguyen, Cao D; Gardiner, Katheleen J; Cios, Krzysztof J

2011-10-01

We introduce a novel method for annotating protein function that combines Naïve Bayes and association rules, and takes advantage of the underlying topology in protein interaction networks and the structure of graphs in the Gene Ontology. We apply our method to proteins from the Human Protein Reference Database (HPRD) and show that, in comparison with other approaches, it predicts protein functions with significantly higher recall with no loss of precision. Specifically, it achieves 51% precision and 60% recall versus 45% and 26% for Majority and 24% and 61% for χ²-statistics, respectively. Copyright © 2011 Elsevier Inc. All rights reserved.

The V0-ATPase mediates apical secretion of exosomes containing Hedgehog-related proteins in Caenorhabditis elegans

PubMed Central

Liégeois, Samuel; Benedetto, Alexandre; Garnier, Jean-Marie; Schwab, Yannick; Labouesse, Michel

2006-01-01

Polarized intracellular trafficking in epithelia is critical in development, immunity, and physiology to deliver morphogens, defensins, or ion pumps to the appropriate membrane domain. The mechanisms that control apical trafficking remain poorly defined. Using Caenorhabditis elegans, we characterize a novel apical secretion pathway involving multivesicularbodies and the release of exosomes at the apical plasma membrane. By means of two different genetic approaches, we show that the membrane-bound V0 sector of the vacuolar H+-ATPase (V-ATPase) acts in this pathway, independent of its contribution to the V-ATPase proton pump activity. Specifically, we identified mutations in the V0 “a” subunit VHA-5 that affect either the V0-specific function or the V0+V1 function of the V-ATPase. These mutations allowed us to establish that the V0 sector mediates secretion of Hedgehog-related proteins. Our data raise the possibility that the V0 sector mediates exosome and morphogen release in mammals. PMID:16785323

HIF-1 maintains a functional relationship between pancreatic cancer cells and stromal fibroblasts by upregulating expression and secretion of Sonic hedgehog

PubMed Central

Katagiri, Tomohiro; Kobayashi, Minoru; Yoshimura, Michio; Morinibu, Akiyo; Itasaka, Satoshi; Hiraoka, Masahiro; Harada, Hiroshi

2018-01-01

Hypoxic and stroma-rich microenvironments, characteristic features of pancreatic cancers, are strongly associated with a poor prognosis. However, whether and how hypoxia increases stromal compartments remain largely unknown. Here, we investigated the potential importance of a master regulator of the cellular adaptive response to hypoxia, hypoxia-inducible factor-1 (HIF-1), in the formation of stroma-rich microenvironments of pancreatic tumors. We found that pancreatic cancer cells secreted more Sonic hedgehog protein (SHH) under hypoxia by upregulating its expression and efficiency of secretion in a HIF-1-dependent manner. Recombinant SHH, which was confirmed to activate the hedgehog signaling pathway, accelerated the growth of fibroblasts in a dose-dependent manner. The SHH protein secreted from pancreatic cancer cells under hypoxic conditions promoted the growth of fibroblasts by stimulating their Sonic hedgehog signaling pathway. These results suggest that the increased secretion of SHH by HIF-1 is potentially responsible for the formation of detrimental and stroma-rich microenvironments in pancreatic cancers, therefore providing a rational basis to target it in cancer therapy. PMID:29535824

Construction of reliable protein-protein interaction networks with a new interaction generality measure.

PubMed

Saito, Rintaro; Suzuki, Harukazu; Hayashizaki, Yoshihide

2003-04-12

Recent screening techniques have made large amounts of protein-protein interaction data available, from which biologically important information such as the function of uncharacterized proteins, the existence of novel protein complexes, and novel signal-transduction pathways can be discovered. However, experimental data on protein interactions contain many false positives, making these discoveries difficult. Therefore computational methods of assessing the reliability of each candidate protein-protein interaction are urgently needed. We developed a new 'interaction generality' measure (IG2) to assess the reliability of protein-protein interactions using only the topological properties of their interaction-network structure. Using yeast protein-protein interaction data, we showed that reliable protein-protein interactions had significantly lower IG2 values than less-reliable interactions, suggesting that IG2 values can be used to evaluate and filter interaction data to enable the construction of reliable protein-protein interaction networks.

Interactions between whey proteins and salivary proteins as related to astringency of whey protein beverages at low pH.

PubMed

Ye, A; Streicher, C; Singh, H

2011-12-01

Whey protein beverages have been shown to be astringent at low pH. In the present study, the interactions between model whey proteins (β-lactoglobulin and lactoferrin) and human saliva in the pH range from 7 to 2 were investigated using particle size, turbidity, and ζ-potential measurements and sodium dodecyl sulfate-PAGE. The correlation between the sensory results of astringency and the physicochemical data was discussed. Strong interactions between β-lactoglobulin and salivary proteins led to an increase in the particle size and turbidity of mixtures of both unheated and heated β-lactoglobulin and human saliva at pH ∼3.4. However, the large particle size and high turbidity that occurred at pH 2.0 were the result of aggregation of human salivary proteins. The intense astringency in whey protein beverages may result from these increases in particle size and turbidity at these pH values and from the aggregation and precipitation of human salivary proteins alone at pH <3.0. The involvement of salivary proteins in the interaction is a key factor in the perception of astringency in whey protein beverages. At any pH, the increases in particle size and turbidity were much smaller in mixtures of lactoferrin and saliva, which suggests that aggregation and precipitation may not be the only mechanism linked to the perception of astringency in whey protein. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

The role of nutraceuticals in the regulation of Wnt and Hedgehog signaling in cancer

PubMed Central

Li, Yiwei; Wang, Zhiwei; Kong, Dejuan

2010-01-01

Multiple cellular signaling pathways have been involved in the processes of cancer cell invasion and metastasis. Among many signaling pathways, Wnt and Hedgehog (Hh) signaling pathways are critically involved in embryonic development, in the biology of cancer stem cells (CSCs) and in the acquisition of epithelial to mesenchymal transition (EMT), and thus this article will remain focused on Wnt and Hh signaling. Since CSCs and EMT are also known to be responsible for cancer cell invasion and metastasis, the Wnt and Hedgehog signaling pathways are also intimately associated with cancer invasion and metastasis. Emerging evidence suggests the beneficial role of chemopreventive agents commonly known as nutraceutical in cancer. Among many such agents, soy isoflavones, curcumin, green tea polyphenols, 3,3′-diindolylmethane, resveratrol, lycopene, vitamin D, etc. have been found to prevent, reverse, or delay the carcinogenic process. Interestingly, these agents have also shown to prevent or delay the progression of cancer, which could in part be due to their ability to attack CSCs or EMT-type cells by attenuating the Wnt and Hedgehog signaling pathways. In this review, we summarize the current state of our knowledge on the role of Wnt and Hedgehog signaling pathways, and their targeted inactivation by chemopreventive agents (nutraceuticals) for the prevention of tumor progression and/or treatment of human malignancies. PMID:20711635

Mechanisms of action of Coxiella burnetii effectors inferred from host-pathogen protein interactions.

PubMed

Wallqvist, Anders; Wang, Hao; Zavaljevski, Nela; Memišević, Vesna; Kwon, Keehwan; Pieper, Rembert; Rajagopala, Seesandra V; Reifman, Jaques

2017-01-01

Coxiella burnetii is an obligate Gram-negative intracellular pathogen and the etiological agent of Q fever. Successful infection requires a functional Type IV secretion system, which translocates more than 100 effector proteins into the host cytosol to establish the infection, restructure the intracellular host environment, and create a parasitophorous vacuole where the replicating bacteria reside. We used yeast two-hybrid (Y2H) screening of 33 selected C. burnetii effectors against whole genome human and murine proteome libraries to generate a map of potential host-pathogen protein-protein interactions (PPIs). We detected 273 unique interactions between 20 pathogen and 247 human proteins, and 157 between 17 pathogen and 137 murine proteins. We used orthology to combine the data and create a single host-pathogen interaction network containing 415 unique interactions between 25 C. burnetii and 363 human proteins. We further performed complementary pairwise Y2H testing of 43 out of 91 C. burnetii-human interactions involving five pathogen proteins. We used the combined data to 1) perform enrichment analyses of target host cellular processes and pathways, 2) examine effectors with known infection phenotypes, and 3) infer potential mechanisms of action for four effectors with uncharacterized functions. The host-pathogen interaction profiles supported known Coxiella phenotypes, such as adapting cell morphology through cytoskeletal re-arrangements, protein processing and trafficking, organelle generation, cholesterol processing, innate immune modulation, and interactions with the ubiquitin and proteasome pathways. The generated dataset of PPIs-the largest collection of unbiased Coxiella host-pathogen interactions to date-represents a rich source of information with respect to secreted pathogen effector proteins and their interactions with human host proteins.

Amyloid precursor protein and Presenilin 1 interaction studied by FRET in human H4 cells.

PubMed

Nizzari, Mario; Venezia, Valentina; Bianchini, Paolo; Caorsi, Valentina; Diaspro, Alberto; Repetto, Emanuela; Thellung, Stefano; Corsaro, Alessandro; Carlo, Pia; Schettini, Gennaro; Florio, Tullio; Russo, Claudio

2007-01-01

The mayor pathologic hallmarks of Alzheimer's disease (AD) are senile plaque and neurofibrillary tangles. Senile plaque are primarily made up of deposits of amyloid-beta protein, a proteolytic product derived from the amyloid precursor protein (APP). APP is a transmembrane protein detected into the endoplasmic reticulum, in the Golgi apparatus, at the cell surface, recycled by endocytosis to endosomes, whose physiological function is unclear. Presenilins (PS), are a component of gamma-secretase complex that cleave alpha-CTFs (carboxy-terminal fragment), or beta-CTFs, leaving 40 or 42 amino acids amyloid-beta peptides and 58 or 56 amino acids intracellular domains (AICD). Where the amyloid-beta peptides is generated is not clear. The study of APP-PS interaction in specific cell compartments provides a good opportunity to light upon the molecular mechanisms regulating the activity of the "gamma-secretase complex," and where beta-amyloid is generated. In our study we used a biophysical assay of protein proximity: fluorescence resonance energy transfer (FRET), that can provide information about molecular interactions when two proteins are in the close proximity (<10 nm), to examine the subcellular localization and interaction between APP and PS1 in human neuroglioma cells (H4). Confocal microscopic analysis reveals extensive colocalization in different cells' compartment, and centrosomal or microtubule organizing center (MTOC) localization of APP and PS1, but not necessarily a close molecular interaction. We used FRET to determine if APP and PS1 interact at the cell centrosome. FRET data suggest a close interaction between APP and PS1 in subcellular compartments and at the centrosome of H4 cells. Using this approach we show that APP and PS1 are closely associated in the centrosomes of the H4 cell.

Spontaneous neoplasia in four captive greater hedgehog tenrecs (Setifer setosus).

PubMed

Khoii, Mina K; Howerth, Elizabeth W; Burns, Roy B; Carmichael, K Paige; Gyimesi, Zoltan S

2008-09-01

Little information is available about diseases and pathology of species within the family Tenrecidae, including the greater hedgehog tenrec (Setifer setosus), a Madagascan insectivore. This report summarizes necropsy and histopathologic findings of neoplasia in four captive greater hedgehog tenrecs. Although only four animals are included in this report, neoplasia seems to be a common and significant source of morbidity and mortality in greater hedgehog tenrecs. Types of neoplasia identified include a thyroid follicular-solid carcinoma, two urinary bladder transitional cell carcinomas, uterine endometrial polyps, and multicentric B-cell lymphoma. Due to small sample size, no etiology could be determined, but genetics, viral infection, pesticide treatment, nutrition, or other environmental factors might contribute to the development of neoplasia in this species. This is the first report of neoplasia in greater hedgehog tenrecs.

Stapled peptides as a new technology to investigate protein-protein interactions in human platelets.

PubMed

Iegre, Jessica; Ahmed, Niaz S; Gaynord, Josephine S; Wu, Yuteng; Herlihy, Kara M; Tan, Yaw Sing; Lopes-Pires, Maria E; Jha, Rupam; Lau, Yu Heng; Sore, Hannah F; Verma, Chandra; O' Donovan, Daniel H; Pugh, Nicholas; Spring, David R

2018-05-28

Platelets are blood cells with numerous crucial pathophysiological roles in hemostasis, cardiovascular thrombotic events and cancer metastasis. Platelet activation requires the engagement of intracellular signalling pathways that involve protein-protein interactions (PPIs). A better understanding of these pathways is therefore crucial for the development of selective anti-platelet drugs. New strategies for studying PPIs in human platelets are required to overcome limitations associated with conventional platelet research methods. For example, small molecule inhibitors can lack selectivity and are often difficult to design and synthesise. Additionally, development of transgenic animal models is costly and time-consuming and conventional recombinant techniques are ineffective due to the lack of a nucleus in platelets. Herein, we describe the generation of a library of novel, functionalised stapled peptides and their first application in the investigation of platelet PPIs. Moreover, the use of platelet-permeable stapled Bim BH3 peptides confirms the part of Bim in phosphatidyl-serine (PS) exposure and reveals a role for the Bim protein in platelet activatory processes. Our work demonstrates that functionalised stapled peptides are a complementary alternative to conventional platelet research methods, and could make a significant contribution to the understanding of platelet signalling pathways and hence to the development of anti-platelet drugs.

Indian hedgehog signaling triggers Nkx3.2 protein degradation during chondrocyte maturation

PubMed Central

Choi, Seung-Won; Jeong, Da-Un; Kim, Jeong-Ah; Lee, Boyoung; Joeng, Kyu Sang; Long, Fanxin; Kim, Dae-Won

2015-01-01

The Indian hedgehog (Ihh) pathway plays an essential role in facilitating chondrocyte hypertrophy and bone formation during skeletal development. Nkx3.2 is initially induced in chondrocyte precursor cells, maintained in early-stage chondrocytes, and down-regulated in terminal-stage chondrocytes. Consistent with these expression patterns, Nkx3.2 has been shown to enhance chondrocyte differentiation and cell survival, while inhibiting chondrocyte hypertrophy and apoptosis. Thus, in this work, we investigate whether Nkx3.2, an early stage chondrogenic factor, can be regulated by Ihh, a key regulator for chondrocyte hypertrophy. Here, we show that Ihh signaling can induce proteasomal degradation of Nkx3.2. In addition, we found that Ihh can suppress levels of Lrp (Wnt co-receptor) and Sfrp (Wnt antagonist) expression, which, in turn, may selectively enhance Lrp-independent non-canonical Wnt pathways in chondrocyte. In agreement with these findings, Ihh-induced Nkx3.2 degradation requires Wnt5a, which is capable of triggering Nkx3.2 degradation. Finally, we found that Nkx3.2 protein levels in chondrocytes are remarkably elevated in mice defective in Ihh signaling by deletion of either Ihh or Smoothened. Thus, these results suggest that Ihh/Wnt5a signaling may play a role in negative regulation of Nkx3.2 for appropriate progression of chondrocyte hypertrophy during chondrogenesis. PMID:22507129

Molecular imaging of drug-modulated protein-protein interactions in living subjects.

PubMed

Paulmurugan, Ramasamy; Massoud, Tarik F; Huang, Jing; Gambhir, Sanjiv S

2004-03-15

Networks of protein interactions mediate cellular responses to environmental stimuli and direct the execution of many different cellular functional pathways. Small molecules synthesized within cells or recruited from the external environment mediate many protein interactions. The study of small molecule-mediated interactions of proteins is important to understand abnormal signal transduction pathways in cancer and in drug development and validation. In this study, we used split synthetic renilla luciferase (hRLUC) protein fragment-assisted complementation to evaluate heterodimerization of the human proteins FRB and FKBP12 mediated by the small molecule rapamycin. The concentration of rapamycin required for efficient dimerization and that of its competitive binder ascomycin required for dimerization inhibition were studied in cell lines. The system was dually modulated in cell culture at the transcription level, by controlling nuclear factor kappaB promoter/enhancer elements using tumor necrosis factor alpha, and at the interaction level, by controlling the concentration of the dimerizer rapamycin. The rapamycin-mediated dimerization of FRB and FKBP12 also was studied in living mice by locating, quantifying, and timing the hRLUC complementation-based bioluminescence imaging signal using a cooled charged coupled device camera. This split reporter system can be used to efficiently screen small molecule drugs that modulate protein-protein interactions and also to assess drugs in living animals. Both are essential steps in the preclinical evaluation of candidate pharmaceutical agents targeting protein-protein interactions, including signaling pathways in cancer cells.

Integrin-associated protein (CD47) is a putative mediator for soluble fibrinogen interaction with human red blood cells membrane.

PubMed

De Oliveira, S; Vitorino de Almeida, V; Calado, A; Rosário, H S; Saldanha, C

2012-03-01

Fibrinogen is a multifunctional plasma protein that plays a crucial role in several biological processes. Elevated fibrinogen induces erythrocyte hyperaggregation, suggesting an interaction between this protein and red blood cells (RBCs). Several studies support the concept that fibrinogen interacts with RBC membrane and this binding, due to specific and non-specific mechanisms, may be a trigger to RBC hyperaggregation in inflammation. The main goals of our work were to prove that human RBCs are able to specifically bind soluble fibrinogen, and identify membrane molecular targets that could be involved in this process. RBCs were first isolated from blood of healthy individuals and then separated in different age fractions by discontinuous Percoll gradients. After isolation RBC samples were incubated with human soluble fibrinogen and/or with a blocking antibody against CD47 followed by fluorescence confocal microscopy, flow cytometry acquisitions and zeta potential measurements. Our data show that soluble fibrinogen interacts with the human RBC membrane in an age-dependent manner, with younger RBCs interacting more with soluble fibrinogen than the older cells. Importantly, this interaction is abrogated in the presence of a specific antibody against CD47. Our results support a specific and age-dependent interaction of soluble fibrinogen with human RBC membrane; additionally we present CD47 as a putative mediator in this process. This interaction may contribute to RBC hyperaggregation in inflammation. Copyright © 2011 Elsevier B.V. All rights reserved.

Characterization of the interactions between protein and carbon black.

PubMed

Chen, Tzu-Tao; Chuang, Kai-Jen; Chiang, Ling-Ling; Chen, Chun-Chao; Yeh, Chi-Tai; Wang, Liang-Shun; Gregory, Clive; Jones, Tim; BéruBé, Kelly; Lee, Chun-Nin; Chuang, Hsiao-Chi; Cheng, Tsun-Jen

2014-01-15

A considerable amount of studies have been conducted to investigate the interactions of biological fluids with nanoparticle surfaces, which exhibit a high affinity for proteins and particles. However, the mechanisms underlying these interactions have not been elucidated, particularly as they relate to human health. Using bovine serum albumin (BSA) and mice bronchoalveolar lavage fluid (BALF) as models for protein-particle conjugates, we characterized the physicochemical modifications of carbon blacks (CB) with 23nm or 65nm in diameter after protein treatment. Adsorbed BALF-containing proteins were quantified and identified by pathways, biological analyses and protein classification. Significant modifications of the physicochemistry of CB were induced by the addition of BSA. Enzyme modulators and hydrolase predominately interacted with CB, with protein-to-CB interactions that were associated with the coagulation pathways. Additionally, our results revealed that an acute-phase response could be activated by these proteins. With regard to human health, the present study revealed that the CB can react with proteins (∼55kDa and 70kDa) after inhalation and may modify the functional structures of lung proteins, leading to the activation of acute-inflammatory responses in the lungs. Copyright © 2013 Elsevier B.V. All rights reserved.

Notochord-derived hedgehog is essential for tail regeneration in Xenopus tadpole.

PubMed

Taniguchi, Yuka; Watanabe, Kenji; Mochii, Makoto

2014-06-18

Appendage regeneration in amphibians is regulated by the combinatorial actions of signaling molecules. The requirement of molecules secreted from specific tissues is reflected by the observation that the whole process of regeneration can be inhibited if a certain tissue is removed from the amputated stump. Interestingly, urodeles and anurans show different tissue dependencies during tail regeneration. The spinal cord is essential for tail regeneration in urodele but not in anuran larva, whereas the notochord but not the spinal cord is essential for tail regeneration in anuran tadpoles. Sonic hedgehog is one of the signaling molecules responsible for such phenomenon in axolotl, as hedgehog signaling is essential for overall tail regeneration and sonic hedgehog is exclusively expressed in the spinal cord. In order to know whether hedgehog signaling is involved in the molecular mechanism underlying the inconsistent tissue dependency for tail regeneration between anurans and urodeles, we investigated expression of hedgehog signal-related genes in the regenerating tail of Xenopus tadpole and examined the effect of the hedgehog signal inhibitor, cyclopamine, on the tail regeneration. In Xenopus, sonic hedgehog is expressed exclusively in the notochord but not in the spinal cord of the regenerate. Overall regeneration was severely impaired in cyclopamine-treated tadpoles. Notochord maturation in the regenerate, including cell alignment and vacuolation, and myofiber formation were inhibited. Proliferation of spinal cord cells in the neural ampulla and of mesenchymal cells was also impaired. As in the axolotl, hedgehog signaling is required for multiple steps in tail regeneration in the Xenopus tadpole, although the location of the Shh source is quite different between the two species. This difference in Shh localization is the likely basis for the differing tissue requirement for tail regeneration between urodeles and anurans.

Notochord-derived hedgehog is essential for tail regeneration in Xenopus tadpole

PubMed Central

2014-01-01

Background Appendage regeneration in amphibians is regulated by the combinatorial actions of signaling molecules. The requirement of molecules secreted from specific tissues is reflected by the observation that the whole process of regeneration can be inhibited if a certain tissue is removed from the amputated stump. Interestingly, urodeles and anurans show different tissue dependencies during tail regeneration. The spinal cord is essential for tail regeneration in urodele but not in anuran larva, whereas the notochord but not the spinal cord is essential for tail regeneration in anuran tadpoles. Sonic hedgehog is one of the signaling molecules responsible for such phenomenon in axolotl, as hedgehog signaling is essential for overall tail regeneration and sonic hedgehog is exclusively expressed in the spinal cord. In order to know whether hedgehog signaling is involved in the molecular mechanism underlying the inconsistent tissue dependency for tail regeneration between anurans and urodeles, we investigated expression of hedgehog signal-related genes in the regenerating tail of Xenopus tadpole and examined the effect of the hedgehog signal inhibitor, cyclopamine, on the tail regeneration. Results In Xenopus, sonic hedgehog is expressed exclusively in the notochord but not in the spinal cord of the regenerate. Overall regeneration was severely impaired in cyclopamine-treated tadpoles. Notochord maturation in the regenerate, including cell alignment and vacuolation, and myofiber formation were inhibited. Proliferation of spinal cord cells in the neural ampulla and of mesenchymal cells was also impaired. Conclusion As in the axolotl, hedgehog signaling is required for multiple steps in tail regeneration in the Xenopus tadpole, although the location of the Shh source is quite different between the two species. This difference in Shh localization is the likely basis for the differing tissue requirement for tail regeneration between urodeles and anurans. PMID:24941877

Administration of sonic hedgehog protein induces angiogenesis and has therapeutic effects after stroke in rats.

PubMed

Chen, Sheng-Cai; Huang, Ming; He, Quan-Wei; Zhang, Yan; Opoku, Elvis Nana; Yang, Hang; Jin, Hui-Juan; Xia, Yuan-Peng; Hu, Bo

2017-06-03

The Sonic hedgehog (Shh) signaling pathway is recapitulated in response to ischemic injury. Here, we investigated the clinical implications of Shh protein in the ischemic stroke and explored the underlying mechanism. Intracerebroventricular injection of Shh, Cyclopamine, or anti-vascular endothelial growth factor (VEGF) was performed immediately after permanent middle cerebral artery occlusion (pMCAO) surgery and lasted for 7days (d). Phosphate-buffered saline (PBS) was used as control. Neurological deficits and infarct volume were examined 7d after pMCAO. Microvascular density with fluorescein-iso-thiocyanate (FITC) assay and double staining with CD31 and Ki-67 was measured at 7d. To observe in vitro angiogenesis, rat brain microvascular endothelial cells (RBMECs) were incubated under oxygen glucose deprivation (OGD) for 6h (h) and treated with Shh/anti-VEGF. We found that (1) Shh improved neurological scores and reduced infarct volume, which was blocked by Cyclopamine, (2) Shh improved the microvascular density and promoted angiogenesis and neuron survival in the ischemic boundary zone, (3) Shh enhanced VEGF expression and VEGF antibody could reverse angiogenic and protective effect of Shh in vivo and in vitro. These data demonstrate that the administration of Shh protein could protect brain from ischemic injury, in part by promoting angiogenic repair. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Interaction entropy for protein-protein binding

NASA Astrophysics Data System (ADS)

Sun, Zhaoxi; Yan, Yu N.; Yang, Maoyou; Zhang, John Z. H.

2017-03-01

Protein-protein interactions are at the heart of signal transduction and are central to the function of protein machine in biology. The highly specific protein-protein binding is quantitatively characterized by the binding free energy whose accurate calculation from the first principle is a grand challenge in computational biology. In this paper, we show how the interaction entropy approach, which was recently proposed for protein-ligand binding free energy calculation, can be applied to computing the entropic contribution to the protein-protein binding free energy. Explicit theoretical derivation of the interaction entropy approach for protein-protein interaction system is given in detail from the basic definition. Extensive computational studies for a dozen realistic protein-protein interaction systems are carried out using the present approach and comparisons of the results for these protein-protein systems with those from the standard normal mode method are presented. Analysis of the present method for application in protein-protein binding as well as the limitation of the method in numerical computation is discussed. Our study and analysis of the results provided useful information for extracting correct entropic contribution in protein-protein binding from molecular dynamics simulations.

Exploring the interactions of the RAS family in the human protein network and their potential implications in RAS-directed therapies

PubMed Central

Bueno, Anibal; Morilla, Ian; Diez, Diego; Moya-Garcia, Aurelio A.; Lozano, José; Ranea, Juan A.G.

2016-01-01

RAS proteins are the founding members of the RAS superfamily of GTPases. They are involved in key signaling pathways regulating essential cellular functions such as cell growth and differentiation. As a result, their deregulation by inactivating mutations often results in aberrant cell proliferation and cancer. With the exception of the relatively well-known KRAS, HRAS and NRAS proteins, little is known about how the interactions of the other RAS human paralogs affect cancer evolution and response to treatment. In this study we performed a comprehensive analysis of the relationship between the phylogeny of RAS proteins and their location in the protein interaction network. This analysis was integrated with the structural analysis of conserved positions in available 3D structures of RAS complexes. Our results show that many RAS proteins with divergent sequences are found close together in the human interactome. We found specific conserved amino acid positions in this group that map to the binding sites of RAS with many of their signaling effectors, suggesting that these pairs could share interacting partners. These results underscore the potential relevance of cross-talking in the RAS signaling network, which should be taken into account when considering the inhibitory activity of drugs targeting specific RAS oncoproteins. This study broadens our understanding of the human RAS signaling network and stresses the importance of considering its potential cross-talk in future therapies. PMID:27713118

Viral Interactions with PDZ Domain-Containing Proteins-An Oncogenic Trait?

PubMed

James, Claire D; Roberts, Sally

2016-01-18

Many of the human viruses with oncogenic capabilities, either in their natural host or in experimental systems (hepatitis B and C, human T cell leukaemia virus type 1, Kaposi sarcoma herpesvirus, human immunodeficiency virus, high-risk human papillomaviruses and adenovirus type 9), encode in their limited genome the ability to target cellular proteins containing PSD95/ DLG/ZO-1 (PDZ) interaction modules. In many cases (but not always), the viruses have evolved to bind the PDZ domains using the same short linear peptide motifs found in host protein-PDZ interactions, and in some cases regulate the interactions in a similar fashion by phosphorylation. What is striking is that the diverse viruses target a common subset of PDZ proteins that are intimately involved in controlling cell polarity and the structure and function of intercellular junctions, including tight junctions. Cell polarity is fundamental to the control of cell proliferation and cell survival and disruption of polarity and the signal transduction pathways involved is a key event in tumourigenesis. This review focuses on the oncogenic viruses and the role of targeting PDZ proteins in the virus life cycle and the contribution of virus-PDZ protein interactions to virus-mediated oncogenesis. We highlight how many of the viral associations with PDZ proteins lead to deregulation of PI3K/AKT signalling, benefitting virus replication but as a consequence also contributing to oncogenesis.

Differential Occurrence of Interactions and Interaction Domains in Proteins Containing Homopolymeric Amino Acid Repeats

PubMed Central

Pelassa, Ilaria; Fiumara, Ferdinando

2015-01-01

Homopolymeric amino acids repeats (AARs), which are widespread in proteomes, have often been viewed simply as spacers between protein domains, or even as “junk” sequences with no obvious function but with a potential to cause harm upon expansion as in genetic diseases associated with polyglutamine or polyalanine expansions, including Huntington disease and cleidocranial dysplasia. A growing body of evidence indicates however that at least some AARs can form organized, functional protein structures, and can regulate protein function. In particular, certain AARs can mediate protein-protein interactions, either through homotypic AAR-AAR contacts or through heterotypic contacts with other protein domains. It is still unclear however, whether AARs may have a generalized, proteome-wide role in shaping protein-protein interaction networks. Therefore, we have undertaken here a bioinformatics screening of the human proteome and interactome in search of quantitative evidence of such a role. We first identified the sets of proteins that contain repeats of any one of the 20 amino acids, as well as control sets of proteins chosen at random in the proteome. We then analyzed the connectivity between the proteins of the AAR-containing protein sets and we compared it with that observed in the corresponding control networks. We find evidence for different degrees of connectivity in the different AAR-containing protein networks. Indeed, networks of proteins containing polyglutamine, polyglutamate, polyproline, and other AARs show significantly increased levels of connectivity, whereas networks containing polyleucine and other hydrophobic repeats show lower degrees of connectivity. Furthermore, we observed that numerous protein-protein, -nucleic acid, and -lipid interaction domains are significantly enriched in specific AAR protein groups. These findings support the notion of a generalized, combinatorial role of AARs, together with conventional protein interaction domains, in

The C8-binding protein of human erythrocytes: interaction with the components of the complement-attack phase.

PubMed Central

Schönermark, S; Filsinger, S; Berger, B; Hänsch, G M

1988-01-01

C8-binding protein is an intrinsic membrane protein of the human erythrocyte. It inhibits the complement (C5b-9)-mediated lysis in a species-restricting manner. In the present study we incorporated C8bp, isolated from human erythrocytes, into sheep erythrocytes (SRBC). SRBC, normally sensitive to lysis by human C5b-9, became insensitive to lysis. Furthermore, we found that C8bp is incorporated into the membrane-attack complex C5b-9, most probably by interacting with C8, since C8bp has an affinity for C8, particularly for the C8 alpha-gamma-subunit. Antibodies to C8bp react with the C8 alpha-subunits and with C9, pointing to the possibility of a partial homology between these proteins. Images Figure 4 Figure 6 Figure 7 PMID:3366469

Dissecting protein:protein interactions between transcription factors with an RNA aptamer.

PubMed Central

Tian, Y; Adya, N; Wagner, S; Giam, C Z; Green, M R; Ellington, A D

1995-01-01

Nucleic acid aptamers isolated from random sequence pools have generally proven useful at inhibiting the interactions of nucleic acid binding proteins with their cognate nucleic acids. In order to develop reagents that could also be used to study protein:protein interactions, we have used in vitro selection to search for RNA aptamers that could interact with the transactivating protein Tax from human T-cell leukemia virus. Tax does not normally bind to nucleic acids, but instead stimulates transcription by interacting with a variety of cellular transcription factors, including the cyclic AMP-response element binding protein (CREB), NF-kappa B, and the serum response factor (SRF). Starting from a pool of greater than 10(13) different RNAs with a core of 120 random sequence positions, RNAs were selected for their ability to be co-retained on nitrocellulose filters with Tax. After five cycles of selection and amplification, a single nucleic acid species remained. This aptamer was found to bind Tax with high affinity and specificity, and could disrupt complex formation between Tax and NF-kappa B, but not with SRF. The differential effects of our aptamer probe on protein:protein interactions suggest a model for how the transcription factor binding sites on the surface of the Tax protein are organized. This model is consistent with data from a variety of other studies. PMID:7489503

Human stanniocalcin-1 interacts with nuclear and cytoplasmic proteins and acts as a SUMO E3 ligase.

PubMed

dos Santos, Marcos Tadeu; Trindade, Daniel Maragno; Gonçalves, Kaliandra de Almeida; Bressan, Gustavo Costa; Anastassopoulos, Filipe; Yunes, José Andres; Kobarg, Jörg

2011-01-01

Human stanniocalcin-1 (STC1) is a glycoprotein that has been implicated in different physiological process, including angiogenesis, apoptosis and carcinogenesis. Here we identified STC1 as a putative molecular marker for the leukemic bone marrow microenvironment and identified new interacting protein partners for STC1. Seven selected interactions retrieved from yeast two-hybrid screens were confirmed by GST-pull down assays in vitro. The N-terminal region was mapped to be the region that mediates the interaction with cytoplasmic, mitochondrial and nuclear proteins. STC1 interacts with SUMO-1 and several proteins that have been shown to be SUMOylated and localized to SUMOylation related nuclear bodies. Although STC1 interacts with SUMO-1 and has a high theoretical prediction score for a SUMOylation site, endogenous co-immunoprecipitation and in vitro SUMOylation assays with the purified recombinant protein could not detect STC1 SUMOylation. However, when we tested STC1 for SUMO E3 ligase activity, we found in an in vitro assay, that it significantly increases the SUMOylation of two other proteins. Confocal microscopic subcellular localization studies using both transfected cells and specific antibodies for endogenous STC1 revealed a cytoplasmic and nuclear deposition, the latter in the form of some specific dot-like substructure resembling SUMOylation related nuclear bodies. Together, these findings suggest a new role for STC1 in SUMOylation pathways, in nuclear bodies.

Interaction of Human Plasma Proteins with Thin Gelatin-Based Hydrogel Films: A QCM-D and ToF-SIMS Study

PubMed Central

2015-01-01

In the fields of surgery and regenerative medicine, it is crucial to understand the interactions of proteins with the biomaterials used as implants. Protein adsorption directly influences cell-material interactions in vivo and, as a result, regulates, for example, cell adhesion on the surface of the implant. Therefore, the development of suitable analytical techniques together with well-defined model systems allowing for the detection, characterization, and quantification of protein adsorbates is essential. In this study, a protocol for the deposition of highly stable, thin gelatin-based films on various substrates has been developed. The hydrogel films were characterized morphologically and chemically. Due to the obtained low thickness of the hydrogel layer, this setup allowed for a quantitative study on the interaction of human proteins (albumin and fibrinogen) with the hydrogel by Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D). This technique enables the determination of adsorbant mass and changes in the shear modulus of the hydrogel layer upon adsorption of human proteins. Furthermore, Secondary Ion Mass Spectrometry and principal component analysis was applied to monitor the changed composition of the topmost adsorbate layer. This approach opens interesting perspectives for a sensitive screening of viscoelastic biomaterials that could be used for regenerative medicine. PMID:24956040

Control of somite patterning by Sonic hedgehog and its downstream signal response genes.

PubMed

Borycki, A G; Mendham, L; Emerson, C P

1998-02-01

In the avian embryo, previous work has demonstrated that the notochord provides inductive signals to activate myoD and pax1 regulatory genes, which are expressed in the dorsal and ventral somite cells that give rise to myotomal and sclerotomal lineages. Here, we present bead implantation and antisense inhibition experiments that show that Sonic hedgehog is both a sufficient and essential notochord signal molecule for myoD and pax1 activation in somites. Furthermore, we show that genes of the Sonic hedgehog signal response pathway, specifically patched, the Sonic hedgehog receptor, and gli and gli2/4, zinc-finger transcription factors, are activated in coordination with somite formation, establishing that Sonic hedgehog response genes play a regulatory role in coordinating the response of somites to the constitutive notochord Sonic hedgehog signal. Furthermore, the expression of patched, gli and gli2/4 is differentially patterned in the somite, providing mechanisms for differentially transducing the Sonic hedgehog signal to the myotomal and sclerotomal lineages. Finally, we show that the activation of gli2/4 is controlled by the process of somite formation and signals from the surface ectoderm, whereas upregulation of patched and activation of gli is controlled by the process of somite formation and a Sonic hedgehog signal. The Sonic hedgehog signal response genes, therefore, have important functions in regulating the initiation of the Sonic hedgehog response in newly forming somites and in regulating the patterned expression of myoD and pax1 in the myotomal and sclerotomal lineages following somite formation.

Molecular Modeling of Structures and Interaction of Human Corticotropin-Releasing Factor (CRF) Binding Protein and CRF Type-2 Receptor

PubMed Central

Slater, Paula G.; Gutierrez-Maldonado, Sebastian E.; Gysling, Katia; Lagos, Carlos F.

2018-01-01

The corticotropin-releasing factor (CRF) system is a key mediator of the stress response and addictive behavior. The CRF system includes four peptides: The CRF system includes four peptides: CRF, urocortins I–III, CRF binding protein (CRF-BP) that binds CRF with high affinity, and two class B G-protein coupled receptors CRF1R and CRF2R. CRF-BP is a secreted protein without significant sequence homology to CRF receptors or to any other known class of protein. Recently, it has been described a potentiation role of CRF-BP over CRF signaling through CRF2R in addictive-related neuronal plasticity and behavior. In addition, it has been described that CRF-BP is capable to physically interact specifically with the α isoform of CRF2R and acts like an escort protein increasing the amount of the receptor in the plasma membrane. At present, there are no available structures for CRF-BP or for full-length CRFR. Knowing and studying the structure of these proteins could be beneficial in order to characterize the CRF-BP/CRF2αR interaction. In this work, we report the modeling of CRF-BP and of full-length CRF2αR and CRF2βR based on the recently solved crystal structures of the transmembrane domains of the human glucagon receptor and human CRF1R, in addition with the resolved N-terminal extracellular domain of CRFRs. These models were further studied using molecular dynamics simulations and protein–protein docking. The results predicted a higher possibility of interaction of CRF-BP with CRF2αR than CRF2βR and yielded the possible residues conforming the interacting interface. Thus, the present study provides a framework for further investigation of the CRF-BP/CRF2αR interaction. PMID:29515519

Reduced primary cilia length and altered Arl13b expression are associated with deregulated chondrocyte Hedgehog signaling in alkaptonuria

PubMed Central

Thorpe, Stephen D.; Gambassi, Silvia; Thompson, Clare L.; Chandrakumar, Charmilie

2017-01-01

Alkaptonuria (AKU) is a rare inherited disease resulting from a deficiency of the enzyme homogentisate 1,2‐dioxygenase which leads to the accumulation of homogentisic acid (HGA). AKU is characterized by severe cartilage degeneration, similar to that observed in osteoarthritis. Previous studies suggest that AKU is associated with alterations in cytoskeletal organization which could modulate primary cilia structure/function. This study investigated whether AKU is associated with changes in chondrocyte primary cilia and associated Hedgehog signaling which mediates cartilage degradation in osteoarthritis. Human articular chondrocytes were obtained from healthy and AKU donors. Additionally, healthy chondrocytes were treated with HGA to replicate AKU pathology (+HGA). Diseased cells exhibited shorter cilia with length reductions of 36% and 16% in AKU and +HGA chondrocytes respectively, when compared to healthy controls. Both AKU and +HGA chondrocytes demonstrated disruption of the usual cilia length regulation by actin contractility. Furthermore, the proportion of cilia with axoneme breaks and bulbous tips was increased in AKU chondrocytes consistent with defective regulation of ciliary trafficking. Distribution of the Hedgehog‐related protein Arl13b along the ciliary axoneme was altered such that its localization was increased at the distal tip in AKU and +HGA chondrocytes. These changes in cilia structure/trafficking in AKU and +HGA chondrocytes were associated with a complete inability to activate Hedgehog signaling in response to exogenous ligand. Thus, we suggest that altered responsiveness to Hedgehog, as a consequence of cilia dysfunction, may be a contributing factor in the development of arthropathy highlighting the cilium as a novel target in AKU. PMID:28158906

Hematologic and biochemical variables of hedgehogs (Erinaceus europaeus) after overwintering in rehabilitation centers.

PubMed

Rossi, Gabriele; Mangiagalli, Gerard; Paracchini, Giulia; Paltrinieri, Saverio

2014-03-01

Information about laboratory reference intervals (RIs) of European Hedgehog (Erinaceus europaeus) hospitalized at rehabilitation centers is scarce. The purpose of this study was to establish hematologic and biochemical RIs for rehabilitated hedgehogs before the release into the wild, and to assess whether sex and management of the center influence laboratory results. Blood was collected from 50 hedgehogs at 3 centers. Thirty-eight animals were included in the study based on normal body weight, absence of clinical signs of disease, Bunnell index > 0.80, and absence of hibernation during overwintering. CBCs were performed using an automated laser cell counter followed by morphologic analysis of blood smears. Clinical biochemistry was performed using an automated spectrophotometer. RIs were determined as recommended by the ASVCP guidelines. Hematology profiles revealed a prevalence of lymphocytes, a constant presence of nucleated RBCs, Howell-Jolly bodies and basophils, and bilobed nuclei in neutrophils and eosinophils. Biochemistry profiles were characterized by higher creatinine and urea concentrations, and higher ALP and GGT activities compared with other domestic species. The sex did not influence the results. Conversely, numbers of eosinophils, activated and large granular lymphocytes, and concentrations of total protein, glucose and cholesterol were different among the centers, likely due to different management practices (eg, antiparasitic treatments, environmental exposure to microorganisms, diet). The RIs established in this study can be used to monitor the health status of hedgehogs in rehabilitation centers. As management practices appeared to influence some variables, it is recommended to standardize the management protocols to minimize their influence on laboratory data. © 2014 American Society for Veterinary Clinical Pathology and European Society for Veterinary Clinical Pathology.

Zoonoses of ferrets, hedgehogs, and sugar gliders.

PubMed

Pignon, Charly; Mayer, Jörg

2011-09-01

With urbanization, people live in close proximity to their pets. People often share their living quarters and furniture, and this proximity carries a new potential for pathogen transmission. In addition to the change in lifestyle with our pets, new exotic pets are being introduced to the pet industry regularly. Often, we are unfamiliar with specific clinical signs of diseases in these new exotic pets or the routes of transmission of pathogens for the particular species. This article reviews zoonoses that occur naturally in ferrets, hedgehogs, and sugar gliders, discussing the occurrence and clinical symptoms of these diseases in humans.

Characterization of a novel human sperm-associated antigen 9 (SPAG9) having structural homology with c-Jun N-terminal kinase-interacting protein

PubMed Central

Jagadish, Nirmala; Rana, Ritu; Selvi, Ramasamy; Mishra, Deepshikha; Garg, Manoj; Yadav, Shikha; Herr, John C.; Okumura, Katsuzumi; Hasegawa, Akiko; Koyama, Koji; Suri, Anil

2005-01-01

We report a novel SPAG9 (sperm-associated antigen 9) protein having structural homology with JNK (c-Jun N-terminal kinase)-interacting protein 3. SPAG9, a single copy gene mapped to the human chromosome 17q21.33 syntenic with location of mouse chromosome 11, was earlier shown to be expressed exclusively in testis [Shankar, Mohapatra and Suri (1998) Biochem. Biophys. Res. Commun. 243, 561–565]. The SPAG9 amino acid sequence analysis revealed identity with the JNK-binding domain and predicted coiled-coil, leucine zipper and transmembrane domains. The secondary structure analysis predicted an α-helical structure for SPAG9 that was confirmed by CD spectra. Microsequencing of higher-order aggregates of recombinant SPAG9 by tandem MS confirmed the amino acid sequence and mono atomic mass of 83.9 kDa. Transient expression of SPAG9 and its deletion mutants revealed that both leucine zipper with extended coiled-coil domains and transmembrane domain of SPAG9 were essential for dimerization and proper localization. Studies of MAPK (mitogenactivated protein kinase) interactions demonstrated that SPAG9 interacted with higher binding affinity to JNK3 and JNK2 compared with JNK1. No interaction was observed with p38α or extracellular-signal-regulated kinase pathways. Polyclonal antibodies raised against recombinant SPAG9 recognized native protein in human sperm extracts and localized specifically on the acrosomal compartment of intact human spermatozoa. Acrosome-reacted spermatozoa demonstrated SPAG9 immunofluorescence, indicating its retention on the equatorial segment after the acrosome reaction. Further, anti-SPAG9 antibodies inhibited the binding of human spermatozoa to intact human oocytes as well as to matched hemizona. This is the first report of sperm-associated JNK-binding protein that may have a role in spermatozoa–egg interaction. PMID:15693750

Characterization of a novel human sperm-associated antigen 9 (SPAG9) having structural homology with c-Jun N-terminal kinase-interacting protein.

PubMed

Jagadish, Nirmala; Rana, Ritu; Selvi, Ramasamy; Mishra, Deepshikha; Garg, Manoj; Yadav, Shikha; Herr, John C; Okumura, Katsuzumi; Hasegawa, Akiko; Koyama, Koji; Suri, Anil

2005-07-01

We report a novel SPAG9 (sperm-associated antigen 9) protein having structural homology with JNK (c-Jun N-terminal kinase)-interacting protein 3. SPAG9, a single copy gene mapped to the human chromosome 17q21.33 syntenic with location of mouse chromosome 11, was earlier shown to be expressed exclusively in testis [Shankar, Mohapatra and Suri (1998) Biochem. Biophys. Res. Commun. 243, 561-565]. The SPAG9 amino acid sequence analysis revealed identity with the JNK-binding domain and predicted coiled-coil, leucine zipper and transmembrane domains. The secondary structure analysis predicted an alpha-helical structure for SPAG9 that was confirmed by CD spectra. Microsequencing of higher-order aggregates of recombinant SPAG9 by tandem MS confirmed the amino acid sequence and mono atomic mass of 83.9 kDa. Transient expression of SPAG9 and its deletion mutants revealed that both leucine zipper with extended coiled-coil domains and transmembrane domain of SPAG9 were essential for dimerization and proper localization. Studies of MAPK (mitogenactivated protein kinase) interactions demonstrated that SPAG9 interacted with higher binding affinity to JNK3 and JNK2 compared with JNK1. No interaction was observed with p38alpha or extracellular-signal-regulated kinase pathways. Polyclonal antibodies raised against recombinant SPAG9 recognized native protein in human sperm extracts and localized specifically on the acrosomal compartment of intact human spermatozoa. Acrosome-reacted spermatozoa demonstrated SPAG9 immunofluorescence, indicating its retention on the equatorial segment after the acrosome reaction. Further, anti-SPAG9 antibodies inhibited the binding of human spermatozoa to intact human oocytes as well as to matched hemizona. This is the first report of sperm-associated JNK-binding protein that may have a role in spermatozoa-egg interaction.

Desmoplastic ganglioglioma of the spinal cord in a western European hedgehog (Erinaceus europaeus).

PubMed

Ulrich, Reiner; Stan, Alexandru C; Fehr, Michael; Mallig, Carolin; Puff, Christina

2010-11-01

Gangliogliomas are composed of neoplastic glial and neuronal cells and are extremely rare tumors of the central nervous system of domestic animals. The present report describes the clinical presentation and the pathomorphological and immunophenotypical characteristics of a desmoplastic ganglioglioma in the spinal cord of a 3-year-old male western European hedgehog (Erinaceus europaeus). Clinically, the hedgehog exhibited a skin wound and therapy-resistant paresis of the left hind limb. Necropsy showed dilatation of the urinary bladder. Histologic examination of the thoracic spinal cord revealed a focally extensive infiltrative mass, which consisted of multiple nodules of smaller bipolar or oligopolar glial cells and variably sized polygonal, ganglionic, neuron-like cells embedded in variable amounts of microcystic neuropilic matrix. An area of spindle-shaped cells arranged in interwoven fascicles and surrounded by a prominent network of reticulin fibers was interpreted as desmoplastic leptomeningeal stroma. Immunohistochemistry revealed a moderate number of glial fibrillary acidic protein and S-100-positive cells and processes. In addition, the ganglionic neuron-like cells expressed neurofilament, microtubule-associated protein-2, and neuron-specific enolase. In summary, this spinal cord tumor was composed of astroglial and neuronal cellular elements, justifying the diagnosis of a desmoplastic ganglioglioma.

Detection of Protein Complexes Based on Penalized Matrix Decomposition in a Sparse Protein⁻Protein Interaction Network.

PubMed

Cao, Buwen; Deng, Shuguang; Qin, Hua; Ding, Pingjian; Chen, Shaopeng; Li, Guanghui

2018-06-15

High-throughput technology has generated large-scale protein interaction data, which is crucial in our understanding of biological organisms. Many complex identification algorithms have been developed to determine protein complexes. However, these methods are only suitable for dense protein interaction networks, because their capabilities decrease rapidly when applied to sparse protein⁻protein interaction (PPI) networks. In this study, based on penalized matrix decomposition ( PMD ), a novel method of penalized matrix decomposition for the identification of protein complexes (i.e., PMD pc ) was developed to detect protein complexes in the human protein interaction network. This method mainly consists of three steps. First, the adjacent matrix of the protein interaction network is normalized. Second, the normalized matrix is decomposed into three factor matrices. The PMD pc method can detect protein complexes in sparse PPI networks by imposing appropriate constraints on factor matrices. Finally, the results of our method are compared with those of other methods in human PPI network. Experimental results show that our method can not only outperform classical algorithms, such as CFinder, ClusterONE, RRW, HC-PIN, and PCE-FR, but can also achieve an ideal overall performance in terms of a composite score consisting of F-measure, accuracy (ACC), and the maximum matching ratio (MMR).

Mapping of interaction domains between human repair proteins ERCC1 and XPF.

PubMed

de Laat, W L; Sijbers, A M; Odijk, H; Jaspers, N G; Hoeijmakers, J H

1998-09-15

ERCC1-XPF is a heterodimeric protein complexinvolved in nucleotide excision repair and recombinational processes. Like its homologous complex in Saccharomyces cerevisiae , Rad10-Rad1, it acts as a structure-specific DNA endonuclease, cleaving at duplex-single-stranded DNA junctions. In repair, ERCC1-XPF and Rad10-Rad1 make an incision on the the 5'-side of the lesion. No humans with a defect in the ERCC1 subunit of this protein complex have been identified and ERCC1-deficient mice suffer from severe developmental problems and signs of premature aging on top of a repair-deficient phenotype. Xeroderma pigmentosum group F patients carry mutations in the XPF subunit and generally show the clinical symptoms of mild DNA repair deficiency. All XP-F patients examined demonstrate reduced levels of XPF and ERCC1 protein, suggesting that proper complex formation is required for stability of the two proteins. To better understand the molecular and clinical consequences of mutations in the ERCC1-XPF complex, we decided to map the interaction domains between the two subunits. The XPF-binding domain comprises C-terminal residues 224-297 of ERCC1. Intriguingly, this domain resides outside the region of homology with its yeast Rad10 counterpart. The ERCC1-binding domain in XPF maps to C-terminal residues 814-905. ERCC1-XPF complex formation is established by a direct interaction between these two binding domains. A mutation from an XP-F patient that alters the ERCC1-binding domain in XPF indeed affects complex formation with ERCC1.

Mapping of interaction domains between human repair proteins ERCC1 and XPF.

PubMed Central

de Laat, W L; Sijbers, A M; Odijk, H; Jaspers, N G; Hoeijmakers, J H

1998-01-01

ERCC1-XPF is a heterodimeric protein complexinvolved in nucleotide excision repair and recombinational processes. Like its homologous complex in Saccharomyces cerevisiae , Rad10-Rad1, it acts as a structure-specific DNA endonuclease, cleaving at duplex-single-stranded DNA junctions. In repair, ERCC1-XPF and Rad10-Rad1 make an incision on the the 5'-side of the lesion. No humans with a defect in the ERCC1 subunit of this protein complex have been identified and ERCC1-deficient mice suffer from severe developmental problems and signs of premature aging on top of a repair-deficient phenotype. Xeroderma pigmentosum group F patients carry mutations in the XPF subunit and generally show the clinical symptoms of mild DNA repair deficiency. All XP-F patients examined demonstrate reduced levels of XPF and ERCC1 protein, suggesting that proper complex formation is required for stability of the two proteins. To better understand the molecular and clinical consequences of mutations in the ERCC1-XPF complex, we decided to map the interaction domains between the two subunits. The XPF-binding domain comprises C-terminal residues 224-297 of ERCC1. Intriguingly, this domain resides outside the region of homology with its yeast Rad10 counterpart. The ERCC1-binding domain in XPF maps to C-terminal residues 814-905. ERCC1-XPF complex formation is established by a direct interaction between these two binding domains. A mutation from an XP-F patient that alters the ERCC1-binding domain in XPF indeed affects complex formation with ERCC1. PMID:9722633

Modeling and simulating networks of interdependent protein interactions.

PubMed

Stöcker, Bianca K; Köster, Johannes; Zamir, Eli; Rahmann, Sven

2018-05-21

Protein interactions are fundamental building blocks of biochemical reaction systems underlying cellular functions. The complexity and functionality of these systems emerge not only from the protein interactions themselves but also from the dependencies between these interactions, as generated by allosteric effects or mutual exclusion due to steric hindrance. Therefore, formal models for integrating and utilizing information about interaction dependencies are of high interest. Here, we describe an approach for endowing protein networks with interaction dependencies using propositional logic, thereby obtaining constrained protein interaction networks ("constrained networks"). The construction of these networks is based on public interaction databases as well as text-mined information about interaction dependencies. We present an efficient data structure and algorithm to simulate protein complex formation in constrained networks. The efficiency of the model allows fast simulation and facilitates the analysis of many proteins in large networks. In addition, this approach enables the simulation of perturbation effects, such as knockout of single or multiple proteins and changes of protein concentrations. We illustrate how our model can be used to analyze a constrained human adhesome protein network, which is responsible for the formation of diverse and dynamic cell-matrix adhesion sites. By comparing protein complex formation under known interaction dependencies versus without dependencies, we investigate how these dependencies shape the resulting repertoire of protein complexes. Furthermore, our model enables investigating how the interplay of network topology with interaction dependencies influences the propagation of perturbation effects across a large biochemical system. Our simulation software CPINSim (for Constrained Protein Interaction Network Simulator) is available under the MIT license at http://github.com/BiancaStoecker/cpinsim and as a Bioconda package

The hedgehog system in ovarian follicles of cattle selected for twin ovulations and births: evidence of a link between the IGF and hedgehog systems

USDA-ARS?s Scientific Manuscript database

Hedgehog signaling is involved in regulation of ovarian function in Drosophila but its role in regulating mammalian ovarian folliculogenesis is less clear. Therefore, gene expression of Indian hedgehog (IHH) and its type 1 receptor, patched 1 (PTCH1), were quantified in bovine granulosa (GC) or the...

Macrocyclic peptide inhibitors for the protein-protein interaction of Zaire Ebola virus protein 24 and karyopherin alpha 5.

PubMed

Song, Xiao; Lu, Lu-Yi; Passioura, Toby; Suga, Hiroaki

2017-06-21

Ebola virus infection leads to severe hemorrhagic fever in human and non-human primates with an average case fatality rate of 50%. To date, numerous potential therapies are in development, but FDA-approved drugs or vaccines are yet unavailable. Ebola viral protein 24 (VP24) is a multifunctional protein that plays critical roles in the pathogenesis of Ebola virus infection, e.g. innate immune suppression by blocking the interaction between KPNA and PY-STAT1. Here we report macrocyclic peptide inhibitors of the VP24-KPNA5 protein-protein interaction (PPI) by means of the RaPID (Random non-standard Peptides Integrated Discovery) system. These macrocyclic peptides showed remarkably high affinity to recombinant Zaire Ebola virus VP24 (eVP24), with a dissociation constant in the single digit nanomolar range, and could also successfully disrupt the eVP24-KPNA interaction. This work provides for the first time a chemical probe capable of modulating this PPI interaction and is the starting point for the development of unique anti-viral drugs against the Ebola virus.

Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins

PubMed Central

Steingruber, Mirjam; Kraut, Alexandra; Socher, Eileen; Sticht, Heinrich; Reichel, Anna; Stamminger, Thomas; Amin, Bushra; Couté, Yohann; Hutterer, Corina; Marschall, Manfred

2016-01-01

The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities. PMID:27548200

A Cross-Species Study of PI3K Protein-Protein Interactions Reveals the Direct Interaction of P85 and SHP2

NASA Astrophysics Data System (ADS)

Breitkopf, Susanne B.; Yang, Xuemei; Begley, Michael J.; Kulkarni, Meghana; Chiu, Yu-Hsin; Turke, Alexa B.; Lauriol, Jessica; Yuan, Min; Qi, Jie; Engelman, Jeffrey A.; Hong, Pengyu; Kontaridis, Maria I.; Cantley, Lewis C.; Perrimon, Norbert; Asara, John M.

2016-02-01

Using a series of immunoprecipitation (IP) - tandem mass spectrometry (LC-MS/MS) experiments and reciprocal BLAST, we conducted a fly-human cross-species comparison of the phosphoinositide-3-kinase (PI3K) interactome in a drosophila S2R+ cell line and several NSCLC and human multiple myeloma cell lines to identify conserved interacting proteins to PI3K, a critical signaling regulator of the AKT pathway. Using H929 human cancer cells and drosophila S2R+ cells, our data revealed an unexpected direct binding of Corkscrew, the drosophila ortholog of the non-receptor protein tyrosine phosphatase type II (SHP2) to the Pi3k21B (p60) regulatory subunit of PI3K (p50/p85 human ortholog) but no association with Pi3k92e, the human ortholog of the p110 catalytic subunit. The p85-SHP2 association was validated in human cell lines, and formed a ternary regulatory complex with GRB2-associated-binding protein 2 (GAB2). Validation experiments with knockdown of GAB2 and Far-Western blots proved the direct interaction of SHP2 with p85, independent of adaptor proteins and transfected FLAG-p85 provided evidence that SHP2 binding on p85 occurred on the SH2 domains. A disruption of the SHP2-p85 complex took place after insulin/IGF1 stimulation or imatinib treatment, suggesting that the direct SHP2-p85 interaction was both independent of AKT activation and positively regulates the ERK signaling pathway.

Hedgehog signaling is required at multiple stages of zebrafish tooth development.

PubMed

Jackman, William R; Yoo, James J; Stock, David W

2010-11-30

The accessibility of the developing zebrafish pharyngeal dentition makes it an advantageous system in which to study many aspects of tooth development from early initiation to late morphogenesis. In mammals, hedgehog signaling is known to be essential for multiple stages of odontogenesis; however, potential roles for the pathway during initiation of tooth development or in later morphogenesis are incompletely understood. We have identified mRNA expression of the hedgehog ligands shha and the receptors ptc1 and ptc2 during zebrafish pharyngeal tooth development. We looked for, but did not detect, tooth germ expression of the other known zebrafish hedgehog ligands shhb, dhh, ihha, or ihhb, suggesting that as in mammals, only Shh participates in zebrafish tooth development. Supporting this idea, we found that morphological and gene expression evidence of tooth initiation is eliminated in shha mutant embryos, and that morpholino antisense oligonucleotide knockdown of shha, but not shhb, function prevents mature tooth formation. Hedgehog pathway inhibition with the antagonist compound cyclopamine affected tooth formation at each stage in which we applied it: arresting development at early stages and disrupting mature tooth morphology when applied later. These results suggest that hedgehog signaling is required continuously during odontogenesis. In contrast, over-expression of shha had no effect on the developing dentition, possibly because shha is normally extensively expressed in the zebrafish pharyngeal region. We have identified previously unknown requirements for hedgehog signaling for early tooth initiation and later morphogenesis. The similarity of our results with data from mouse and other vertebrates suggests that despite gene duplication and changes in the location of where teeth form, the roles of hedgehog signaling in tooth development have been largely conserved during evolution.

Acinic cell carcinoma in an African pygmy hedgehog (Atelerix albiventris).

PubMed

Fukuzawa, Ryuji; Fukuzawa, Kazuhiro; Abe, Hitoshi; Nagai, Toshihiro; Kameyama, Kaori

2004-01-01

A male African pygmy hedgehog (Atelerix albiventris), estimated to be 3 years old, presented with exophthalmos and fixed abduction of the right eye. Radiographic examination revealed a retrobulbar tumor in the right orbital cavity. The mass was surgically resected but recurred 3 months later and the hedgehog died. There was no gross or microscopic evidence of salivary or lacrimal gland involvement of the tumor at surgery or at necropsy. The histopathologic, immunohistochemical, and ultrastructural findings were those of acinic cell carcinoma, the origin of which was unknown. This is the first known case of acinic cell carcinoma in an African hedgehog.

Patterns of HIV-1 Protein Interaction Identify Perturbed Host-Cellular Subsystems

PubMed Central

MacPherson, Jamie I.; Dickerson, Jonathan E.; Pinney, John W.; Robertson, David L.

2010-01-01

Human immunodeficiency virus type 1 (HIV-1) exploits a diverse array of host cell functions in order to replicate. This is mediated through a network of virus-host interactions. A variety of recent studies have catalogued this information. In particular the HIV-1, Human Protein Interaction Database (HHPID) has provided a unique depth of protein interaction detail. However, as a map of HIV-1 infection, the HHPID is problematic, as it contains curation error and redundancy; in addition, it is based on a heterogeneous set of experimental methods. Based on identifying shared patterns of HIV-host interaction, we have developed a novel methodology to delimit the core set of host-cellular functions and their associated perturbation from the HHPID. Initially, using biclustering, we identify 279 significant sets of host proteins that undergo the same types of interaction. The functional cohesiveness of these protein sets was validated using a human protein-protein interaction network, gene ontology annotation and sequence similarity. Next, using a distance measure, we group host protein sets and identify 37 distinct higher-level subsystems. We further demonstrate the biological significance of these subsystems by cross-referencing with global siRNA screens that have been used to detect host factors necessary for HIV-1 replication, and investigate the seemingly small intersect between these data sets. Our results highlight significant host-cell subsystems that are perturbed during the course of HIV-1 infection. Moreover, we characterise the patterns of interaction that contribute to these perturbations. Thus, our work disentangles the complex set of HIV-1-host protein interactions in the HHPID, reconciles these with siRNA screens and provides an accessible and interpretable map of infection. PMID:20686668

Hydrophobic Interaction Chromatography for Bottom-Up Proteomics Analysis of Single Proteins and Protein Complexes.

PubMed

Rackiewicz, Michal; Große-Hovest, Ludger; Alpert, Andrew J; Zarei, Mostafa; Dengjel, Jörn

2017-06-02

Hydrophobic interaction chromatography (HIC) is a robust standard analytical method to purify proteins while preserving their biological activity. It is widely used to study post-translational modifications of proteins and drug-protein interactions. In the current manuscript we employed HIC to separate proteins, followed by bottom-up LC-MS/MS experiments. We used this approach to fractionate antibody species followed by comprehensive peptide mapping as well as to study protein complexes in human cells. HIC-reversed-phase chromatography (RPC)-mass spectrometry (MS) is a powerful alternative to fractionate proteins for bottom-up proteomics experiments making use of their distinct hydrophobic properties.

The imidazopyridine derivative JK184 reveals dual roles for microtubules in Hedgehog signaling.

PubMed

Cupido, Tommaso; Rack, Paul G; Firestone, Ari J; Hyman, Joel M; Han, Kyuho; Sinha, Surajit; Ocasio, Cory A; Chen, James K

2009-01-01

Eradicating hedgehogs: The title molecule has been previously identified as a potent inhibitor of the Hedgehog signaling pathway, which gives embryonic cells information needed to develop properly. This molecule is shown to modulate Hedgehog target gene expression by depolymerizing microtubules, thus revealing dual roles of the cytoskeleton in pathway regulation (see figure).

Assessing reliability of protein-protein interactions by integrative analysis of data in model organisms.

PubMed

Lin, Xiaotong; Liu, Mei; Chen, Xue-wen

2009-04-29

Protein-protein interactions play vital roles in nearly all cellular processes and are involved in the construction of biological pathways such as metabolic and signal transduction pathways. Although large-scale experiments have enabled the discovery of thousands of previously unknown linkages among proteins in many organisms, the high-throughput interaction data is often associated with high error rates. Since protein interaction networks have been utilized in numerous biological inferences, the inclusive experimental errors inevitably affect the quality of such prediction. Thus, it is essential to assess the quality of the protein interaction data. In this paper, a novel Bayesian network-based integrative framework is proposed to assess the reliability of protein-protein interactions. We develop a cross-species in silico model that assigns likelihood scores to individual protein pairs based on the information entirely extracted from model organisms. Our proposed approach integrates multiple microarray datasets and novel features derived from gene ontology. Furthermore, the confidence scores for cross-species protein mappings are explicitly incorporated into our model. Applying our model to predict protein interactions in the human genome, we are able to achieve 80% in sensitivity and 70% in specificity. Finally, we assess the overall quality of the experimentally determined yeast protein-protein interaction dataset. We observe that the more high-throughput experiments confirming an interaction, the higher the likelihood score, which confirms the effectiveness of our approach. This study demonstrates that model organisms certainly provide important information for protein-protein interaction inference and assessment. The proposed method is able to assess not only the overall quality of an interaction dataset, but also the quality of individual protein-protein interactions. We expect the method to continually improve as more high quality interaction data from more

Desert hedgehog is a mediator of demyelination in compression neuropathies.

PubMed

Jung, James; Frump, Derek; Su, Jared; Wang, Weiping; Mozaffar, Tahseen; Gupta, Ranjan

2015-09-01

The secreted protein desert hedgehog (dhh) controls the formation of the nerve perineurium during development and is a key component of Schwann cells that ensures peripheral nerve survival. We postulated that dhh may play a critical role in maintaining myelination and investigated its role in demyelination-induced compression neuropathies by using a post-natal model of a chronic nerve injury in wildtype and dhh(-/-) mice. We evaluated demyelination using electrophysiological, morphological, and molecular approaches. dhh transcripts and protein are down-regulated early after injury in wild-type mice, suggesting an intimate relationship between the hedgehog pathway and demyelination. In dhh(-/-) mice, nerve injury induced more prominent and severe demyelination relative to their wild-type counterparts, suggesting a protective role of dhh. Alterations in nerve fiber characteristics included significant decreases in nerve conduction velocity, increased myelin debris, and substantial decreases in internodal length. Furthermore, in vitro studies showed that dhh blockade via either adenovirus-mediated (shRNA) or pharmacological inhibition both resulted in severe demyelination, which could be rescued by exogenous Dhh. Exogenous Dhh was protective against this demyelination and maintained myelination at baseline levels in a custom in vitro bioreactor to applied biophysical forces to myelinated DRG/Schwann cell co-cultures. Together, these results demonstrate a pivotal role for dhh in maintaining myelination. Furthermore, dhh signaling reveals a potential target for therapeutic intervention to prevent and treat demyelination of peripheral nerves in compression neuropathies. Copyright © 2015 Elsevier Inc. All rights reserved.

The effects of non-synonymous single nucleotide polymorphisms (nsSNPs) on protein-protein interactions.

PubMed

Yates, Christopher M; Sternberg, Michael J E

2013-11-01

Non-synonymous single nucleotide polymorphisms (nsSNPs) are single base changes leading to a change to the amino acid sequence of the encoded protein. Many of these variants are associated with disease, so nsSNPs have been well studied, with studies looking at the effects of nsSNPs on individual proteins, for example, on stability and enzyme active sites. In recent years, the impact of nsSNPs upon protein-protein interactions has also been investigated, giving a greater insight into the mechanisms by which nsSNPs can lead to disease. In this review, we summarize these studies, looking at the various mechanisms by which nsSNPs can affect protein-protein interactions. We focus on structural changes that can impair interaction, changes to disorder, gain of interaction, and post-translational modifications before looking at some examples of nsSNPs at human-pathogen protein-protein interfaces and the analysis of nsSNPs from a network perspective. © 2013.

Sonic hedgehog initiates cochlear hair cell regeneration through downregulation of retinoblastoma protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Na; Department of Otolaryngology and Program in Neuroscience, Harvard Medical School and Eaton Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, MA 02114; Chen, Yan

Highlights: Black-Right-Pointing-Pointer Shh activation in neonatal cochleae enhances sensory cell proliferation. Black-Right-Pointing-Pointer Proliferating supporting cells can transdifferentiate into hair cells. Black-Right-Pointing-Pointer Shh promotes proliferation by transiently modulating pRb activity. Black-Right-Pointing-Pointer Shh inhibits pRb by inhibiting transcription and increasing phosphorylation of pRb. -- Abstract: Cell cycle re-entry by cochlear supporting cells and/or hair cells is considered one of the best approaches for restoring hearing loss as a result of hair cell damage. To identify mechanisms that can be modulated to initiate cell cycle re-entry and hair cell regeneration, we studied the effect of activating the sonic hedgehog (Shh) pathway. We showmore » that Shh signaling in postnatal rat cochleae damaged by neomycin leads to renewed proliferation of supporting cells and hair cells. Further, proliferating supporting cells are likely to transdifferentiate into hair cells. Shh treatment leads to inhibition of retinoblastoma protein (pRb) by increasing phosphorylated pRb and reducing retinoblastoma gene transcription. This results in upregulation of cyclins B1, D2, and D3, and CDK1. These results suggest that Shh signaling induces cell cycle re-entry in cochlear sensory epithelium and the production of new hair cells, in part by attenuating pRb function. This study provides an additional route to modulate pRb function with important implications in mammalian hair cell regeneration.« less

Hedgehog Signaling in the Stomach

PubMed Central

Konstantinou, Daniel; Bertaux-Skeirik, Nina; Zavros, Yana

2016-01-01

The Hedgehog (Hh) signaling pathway not only plays a key part in controlling embryonic development, but in the adult stomach governs important cellular events such as epithelial cell differentiation, proliferation, gastric disease and regeneration. In particular, Sonic Hedgehog (Shh) signaling has been well studied for its role in gastric physiology and pathophysiology. Shh is secreted from the gastric parietal cells and contributes to the regeneration of the epithelium in response to injury, or the development of gastritis during Helicobacter pylori infection. Dysregulation of the Shh signaling pathway leads to the disruption of gastric differentiation, loss of gastric acid secretion and the development of cancer. In this chapter, we will review the most recent findings that reveal the role of Shh as a regulator of gastric physiology, regeneration and disease. PMID:27750091

Recovering Protein-Protein and Domain-Domain Interactions from Aggregation of IP-MS Proteomics of Coregulator Complexes

PubMed Central

Mazloom, Amin R.; Dannenfelser, Ruth; Clark, Neil R.; Grigoryan, Arsen V.; Linder, Kathryn M.; Cardozo, Timothy J.; Bond, Julia C.; Boran, Aislyn D. W.; Iyengar, Ravi; Malovannaya, Anna; Lanz, Rainer B.; Ma'ayan, Avi

2011-01-01

Coregulator proteins (CoRegs) are part of multi-protein complexes that transiently assemble with transcription factors and chromatin modifiers to regulate gene expression. In this study we analyzed data from 3,290 immuno-precipitations (IP) followed by mass spectrometry (MS) applied to human cell lines aimed at identifying CoRegs complexes. Using the semi-quantitative spectral counts, we scored binary protein-protein and domain-domain associations with several equations. Unlike previous applications, our methods scored prey-prey protein-protein interactions regardless of the baits used. We also predicted domain-domain interactions underlying predicted protein-protein interactions. The quality of predicted protein-protein and domain-domain interactions was evaluated using known binary interactions from the literature, whereas one protein-protein interaction, between STRN and CTTNBP2NL, was validated experimentally; and one domain-domain interaction, between the HEAT domain of PPP2R1A and the Pkinase domain of STK25, was validated using molecular docking simulations. The scoring schemes presented here recovered known, and predicted many new, complexes, protein-protein, and domain-domain interactions. The networks that resulted from the predictions are provided as a web-based interactive application at http://maayanlab.net/HT-IP-MS-2-PPI-DDI/. PMID:22219718

Recent Coselection in Human Populations Revealed by Protein–Protein Interaction Network

PubMed Central

Qian, Wei; Zhou, Hang; Tang, Kun

2015-01-01

Genome-wide scans for signals of natural selection in human populations have identified a large number of candidate loci that underlie local adaptations. This is surprising given the relatively short evolutionary time since the divergence of the human population. One hypothesis that has not been formally examined is whether and how the recent human evolution may have been shaped by coselection in the context of complex molecular interactome. In this study, genome-wide signals of selection were scanned in East Asians, Europeans, and Africans using 1000 Genome data, and subsequently mapped onto the protein–protein interaction (PPI) network. We found that the candidate genes of recent positive selection localized significantly closer to each other on the PPI network than expected, revealing substantial clustering of selected genes. Furthermore, gene pairs of shorter PPI network distances showed higher similarities of their recent evolutionary paths than those further apart. Last, subnetworks enriched with recent coselection signals were identified, which are substantially overrepresented in biological pathways related to signal transduction, neurogenesis, and immune function. These results provide the first genome-wide evidence for association of recent selection signals with the PPI network, shedding light on the potential mechanisms of recent coselection in the human genome. PMID:25532814

Resveratrol Downregulates Interleukin-6-Stimulated Sonic Hedgehog Signaling in Human Acute Myeloid Leukemia

PubMed Central

Su, Yu-Chieh; Li, Szu-Chin; Wu, Yin-Chi; Wang, Li-Min; Chao, K. S. Clifford; Liao, Hui-Fen

2013-01-01

IL-6 and sonic hedgehog (Shh) signaling molecules are considered to maintain the growth of cancer stem cells (CSCs). Resveratrol, an important integrant in traditional Chinese medicine, possesses certain antitumor effects. However, the mechanisms on regulating acute myeloid leukemia (AML) are unclear. This study first used human subjects to demonstrate that the plasma levels of IL-6 and IL-1β in AML patients were higher and lower, respectively, than healthy donors. The expression of Shh preproproteins, and C- and N-terminal Shh peptides increased in bone marrow and peripheral blood mononuclear cells isolated from AML patients, and the plasma N-Shh secretion was greater. To further clarify the effect of IL-6 and resveratrol in Shh signaling, human AML HL-60 cells were tested. IL-6 upregulated Shh and Gli-1 expression and was accompanied by an increase of cell viability. Resveratrol significantly decreased CSC-related Shh expression, Gli-1 nuclear translocation, and cell viability in IL-6-treated HL-60 cells and had synergistic effect with Shh inhibitor cyclopamine on inhibiting cell growth. Conclusions. IL-6 stimulated the growth of AML cells through Shh signaling, and this effect might be blocked by resveratrol. Further investigations of Shh as a prognostic marker and resveratrol as a therapeutic drug target to CSCs in AML are surely warranted. PMID:23533494

Signatures of Pleiotropy, Economy and Convergent Evolution in a Domain-Resolved Map of Human–Virus Protein–Protein Interaction Networks

PubMed Central

Garamszegi, Sara; Franzosa, Eric A.; Xia, Yu

2013-01-01

A central challenge in host-pathogen systems biology is the elucidation of general, systems-level principles that distinguish host-pathogen interactions from within-host interactions. Current analyses of host-pathogen and within-host protein-protein interaction networks are largely limited by their resolution, treating proteins as nodes and interactions as edges. Here, we construct a domain-resolved map of human-virus and within-human protein-protein interaction networks by annotating protein interactions with high-coverage, high-accuracy, domain-centric interaction mechanisms: (1) domain-domain interactions, in which a domain in one protein binds to a domain in a second protein, and (2) domain-motif interactions, in which a domain in one protein binds to a short, linear peptide motif in a second protein. Analysis of these domain-resolved networks reveals, for the first time, significant mechanistic differences between virus-human and within-human interactions at the resolution of single domains. While human proteins tend to compete with each other for domain binding sites by means of sequence similarity, viral proteins tend to compete with human proteins for domain binding sites in the absence of sequence similarity. Independent of their previously established preference for targeting human protein hubs, viral proteins also preferentially target human proteins containing linear motif-binding domains. Compared to human proteins, viral proteins participate in more domain-motif interactions, target more unique linear motif-binding domains per residue, and contain more unique linear motifs per residue. Together, these results suggest that viruses surmount genome size constraints by convergently evolving multiple short linear motifs in order to effectively mimic, hijack, and manipulate complex host processes for their survival. Our domain-resolved analyses reveal unique signatures of pleiotropy, economy, and convergent evolution in viral-host interactions that are

HVint: A Strategy for Identifying Novel Protein-Protein Interactions in Herpes Simplex Virus Type 1*

PubMed Central

Hernandez, Anna; Buch, Anna; Sodeik, Beate; Cristea, Ileana Mihaela

2016-01-01

Human herpesviruses are widespread human pathogens with a remarkable impact on worldwide public health. Despite intense decades of research, the molecular details in many aspects of their function remain to be fully characterized. To unravel the details of how these viruses operate, a thorough understanding of the relationships between the involved components is key. Here, we present HVint, a novel protein-protein intraviral interaction resource for herpes simplex virus type 1 (HSV-1) integrating data from five external sources. To assess each interaction, we used a scoring scheme that takes into consideration aspects such as the type of detection method and the number of lines of evidence. The coverage of the initial interactome was further increased using evolutionary information, by importing interactions reported for other human herpesviruses. These latter interactions constitute, therefore, computational predictions for potential novel interactions in HSV-1. An independent experimental analysis was performed to confirm a subset of our predicted interactions. This subset covers proteins that contribute to nuclear egress and primary envelopment events, including VP26, pUL31, pUL40, and the recently characterized pUL32 and pUL21. Our findings support a coordinated crosstalk between VP26 and proteins such as pUL31, pUS9, and the CSVC complex, contributing to the development of a model describing the nuclear egress and primary envelopment pathways of newly synthesized HSV-1 capsids. The results are also consistent with recent findings on the involvement of pUL32 in capsid maturation and early tegumentation events. Further, they open the door to new hypotheses on virus-specific regulators of pUS9-dependent transport. To make this repository of interactions readily accessible for the scientific community, we also developed a user-friendly and interactive web interface. Our approach demonstrates the power of computational predictions to assist in the design of

Calcium-dependent interaction of monomeric S100P protein with serum albumin.

PubMed

Kazakov, Alexei S; Shevelyova, Marina P; Ismailov, Ramis G; Permyakova, Maria E; Litus, Ekaterina A; Permyakov, Eugene A; Permyakov, Sergei E

2018-03-01

S100 proteins are multifunctional (intra/extra)cellular mostly dimeric calcium-binding proteins engaged into numerous diseases. We have found that monomeric recombinant human S100P protein interacts with intact human serum albumin (HSA) in excess of calcium ions with equilibrium dissociation constant of 25-50nM, as evidenced by surface plasmon resonance spectroscopy and fluorescent titration by HSA of S100P labelled by fluorescein isothiocyanate. Calcium removal or S100P dimerization abolish the S100P-HSA interaction. The interaction is selective, since S100P does not bind bovine serum albumin and monomeric human S100B lacks interaction with HSA. In vitro glycation of HSA disables its binding to S100P. The revealed selective and highly specific conformation-dependent interaction between S100P and HSA shows that functional properties of monomeric and dimeric forms of S100 proteins are different, and raises concerns on validity of cell-based assays and animal models used for studies of (patho)physiological roles of extracellular S100 proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

Inflammatory PAF Receptor Signaling Initiates Hedgehog Signaling and Kidney Fibrogenesis During Ethanol Consumption

PubMed Central

Latchoumycandane, Calivarathan; Hanouneh, Mohamad; Nagy, Laura E.; McIntyre, Thomas M.

2015-01-01

Acute inflammation either resolves or proceeds to fibrotic repair that replaces functional tissue. Pro-fibrotic hedgehog signaling and induction of its Gli transcription factor in pericytes induces fibrosis in kidney, but molecular instructions connecting inflammation to fibrosis are opaque. We show acute kidney inflammation resulting from chronic ingestion of the common xenobiotic ethanol initiates Gli1 transcription and hedgehog synthesis in kidney pericytes, and promotes renal fibrosis. Ethanol ingestion stimulated transcription of TGF-ß, collagens I and IV, and alpha-smooth muscle actin with accumulation of these proteins. This was accompanied by deposition of extracellular fibrils. Ethanol catabolism by CYP2E1 in kidney generates local reactive oxygen species that oxidize cellular phospholipids to phospholipid products that activate the Platelet-activating Factor receptor (PTAFR) for inflammatory phospholipids. Genetically deleting this ptafr locus abolished accumulation of mRNA for TGF-ß, collagen IV, and α-smooth muscle actin. Loss of PTAFR also abolished ethanol-stimulated Sonic (Shh) and Indian hedgehog (Ihh) expression, and abolished transcription and accumulation of Gli1. Shh induced in pericytes and Ihh in tubules escaped to urine of ethanol-fed mice. Neutrophil myeloperoxidase (MPO) is required for ethanol-induced kidney inflammation, and Shh was not present in kidney or urine of mpo -/- mice. Shh also was present in urine of patients with acute kidney injury, but not in normal individuals or those with fibrotic liver cirrhosis We conclude neither endogenous PTAFR signaling nor CYP2E1-generated radicals alone are sufficient to initiate hedgehog signaling, but instead PTAFR-dependent neutrophil infiltration with myeloperoxidase activation is necessary to initiate ethanol-induced fibrosis in kidney. We also show fibrogenic mediators escape to urine, defining a new class of urinary mechanistic biomarkers of fibrogenesis for an organ not commonly

Computational prediction of protein-protein interactions in Leishmania predicted proteomes.

PubMed

Rezende, Antonio M; Folador, Edson L; Resende, Daniela de M; Ruiz, Jeronimo C

2012-01-01

The Trypanosomatids parasites Leishmania braziliensis, Leishmania major and Leishmania infantum are important human pathogens. Despite of years of study and genome availability, effective vaccine has not been developed yet, and the chemotherapy is highly toxic. Therefore, it is clear just interdisciplinary integrated studies will have success in trying to search new targets for developing of vaccines and drugs. An essential part of this rationale is related to protein-protein interaction network (PPI) study which can provide a better understanding of complex protein interactions in biological system. Thus, we modeled PPIs for Trypanosomatids through computational methods using sequence comparison against public database of protein or domain interaction for interaction prediction (Interolog Mapping) and developed a dedicated combined system score to address the predictions robustness. The confidence evaluation of network prediction approach was addressed using gold standard positive and negative datasets and the AUC value obtained was 0.94. As result, 39,420, 43,531 and 45,235 interactions were predicted for L. braziliensis, L. major and L. infantum respectively. For each predicted network the top 20 proteins were ranked by MCC topological index. In addition, information related with immunological potential, degree of protein sequence conservation among orthologs and degree of identity compared to proteins of potential parasite hosts was integrated. This information integration provides a better understanding and usefulness of the predicted networks that can be valuable to select new potential biological targets for drug and vaccine development. Network modularity which is a key when one is interested in destabilizing the PPIs for drug or vaccine purposes along with multiple alignments of the predicted PPIs were performed revealing patterns associated with protein turnover. In addition, around 50% of hypothetical protein present in the networks received some degree

Protein-protein interaction inference based on semantic similarity of Gene Ontology terms.

PubMed

Zhang, Shu-Bo; Tang, Qiang-Rong

2016-07-21

Identifying protein-protein interactions is important in molecular biology. Experimental methods to this issue have their limitations, and computational approaches have attracted more and more attentions from the biological community. The semantic similarity derived from the Gene Ontology (GO) annotation has been regarded as one of the most powerful indicators for protein interaction. However, conventional methods based on GO similarity fail to take advantage of the specificity of GO terms in the ontology graph. We proposed a GO-based method to predict protein-protein interaction by integrating different kinds of similarity measures derived from the intrinsic structure of GO graph. We extended five existing methods to derive the semantic similarity measures from the descending part of two GO terms in the GO graph, then adopted a feature integration strategy to combines both the ascending and the descending similarity scores derived from the three sub-ontologies to construct various kinds of features to characterize each protein pair. Support vector machines (SVM) were employed as discriminate classifiers, and five-fold cross validation experiments were conducted on both human and yeast protein-protein interaction datasets to evaluate the performance of different kinds of integrated features, the experimental results suggest the best performance of the feature that combines information from both the ascending and the descending parts of the three ontologies. Our method is appealing for effective prediction of protein-protein interaction. Copyright © 2016 Elsevier Ltd. All rights reserved.

Structure of the SPRY domain of the human RNA helicase DDX1, a putative interaction platform within a DEAD-box protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kellner, Julian N.; Meinhart, Anton, E-mail: anton.meinhart@mpimf-heidelberg.mpg.de

The structure of the SPRY domain of the human RNA helicase DDX1 was determined at 2.0 Å resolution. The SPRY domain provides a putative protein–protein interaction platform within DDX1 that differs from other SPRY domains in its structure and conserved regions. The human RNA helicase DDX1 in the DEAD-box family plays an important role in RNA processing and has been associated with HIV-1 replication and tumour progression. Whereas previously described DEAD-box proteins have a structurally conserved core, DDX1 shows a unique structural feature: a large SPRY-domain insertion in its RecA-like consensus fold. SPRY domains are known to function as protein–proteinmore » interaction platforms. Here, the crystal structure of the SPRY domain of human DDX1 (hDSPRY) is reported at 2.0 Å resolution. The structure reveals two layers of concave, antiparallel β-sheets that stack onto each other and a third β-sheet beneath the β-sandwich. A comparison with SPRY-domain structures from other eukaryotic proteins showed that the general β-sandwich fold is conserved; however, differences were detected in the loop regions, which were identified in other SPRY domains to be essential for interaction with cognate partners. In contrast, in hDSPRY these loop regions are not strictly conserved across species. Interestingly, though, a conserved patch of positive surface charge is found that may replace the connecting loops as a protein–protein interaction surface. The data presented here comprise the first structural information on DDX1 and provide insights into the unique domain architecture of this DEAD-box protein. By providing the structure of a putative interaction domain of DDX1, this work will serve as a basis for further studies of the interaction network within the hetero-oligomeric complexes of DDX1 and of its recruitment to the HIV-1 Rev protein as a viral replication factor.« less

Proteins interacting with cloning scars: a source of false positive protein-protein interactions.

PubMed

Banks, Charles A S; Boanca, Gina; Lee, Zachary T; Florens, Laurence; Washburn, Michael P

2015-02-23

A common approach for exploring the interactome, the network of protein-protein interactions in cells, uses a commercially available ORF library to express affinity tagged bait proteins; these can be expressed in cells and endogenous cellular proteins that copurify with the bait can be identified as putative interacting proteins using mass spectrometry. Control experiments can be used to limit false-positive results, but in many cases, there are still a surprising number of prey proteins that appear to copurify specifically with the bait. Here, we have identified one source of false-positive interactions in such studies. We have found that a combination of: 1) the variable sequence of the C-terminus of the bait with 2) a C-terminal valine "cloning scar" present in a commercially available ORF library, can in some cases create a peptide motif that results in the aberrant co-purification of endogenous cellular proteins. Control experiments may not identify false positives resulting from such artificial motifs, as aberrant binding depends on sequences that vary from one bait to another. It is possible that such cryptic protein binding might occur in other systems using affinity tagged proteins; this study highlights the importance of conducting careful follow-up studies where novel protein-protein interactions are suspected.

Proteins interacting with cloning scars: a source of false positive protein-protein interactions

PubMed Central

Banks, Charles A. S.; Boanca, Gina; Lee, Zachary T.; Florens, Laurence; Washburn, Michael P.

2015-01-01

A common approach for exploring the interactome, the network of protein-protein interactions in cells, uses a commercially available ORF library to express affinity tagged bait proteins; these can be expressed in cells and endogenous cellular proteins that copurify with the bait can be identified as putative interacting proteins using mass spectrometry. Control experiments can be used to limit false-positive results, but in many cases, there are still a surprising number of prey proteins that appear to copurify specifically with the bait. Here, we have identified one source of false-positive interactions in such studies. We have found that a combination of: 1) the variable sequence of the C-terminus of the bait with 2) a C-terminal valine “cloning scar” present in a commercially available ORF library, can in some cases create a peptide motif that results in the aberrant co-purification of endogenous cellular proteins. Control experiments may not identify false positives resulting from such artificial motifs, as aberrant binding depends on sequences that vary from one bait to another. It is possible that such cryptic protein binding might occur in other systems using affinity tagged proteins; this study highlights the importance of conducting careful follow-up studies where novel protein-protein interactions are suspected. PMID:25704442

Construction of high-quality Caco-2 three-frame cDNA library and its application to yeast two-hybrid for the human astrovirus protein-protein interaction.

PubMed

Zhao, Wei; Li, Xin; Liu, Wen-Hui; Zhao, Jian; Jin, Yi-Ming; Sui, Ting-Ting

2014-09-01

Human epithelial colorectal adenocarcinoma (Caco-2) cells are widely used as an in vitro model of the human small intestinal mucosa. Caco-2 cells are host cells of the human astrovirus (HAstV) and other enteroviruses. High quality cDNA libraries are pertinent resources and critical tools for protein-protein interaction research, but are currently unavailable for Caco-2 cells. To construct a three-open reading frame, full length-expression cDNA library from the Caco-2 cell line for application to HAstV protein-protein interaction screening, total RNA was extracted from Caco-2 cells. The switching mechanism at the 5' end of the RNA transcript technique was used for cDNA synthesis. Double-stranded cDNA was digested by Sfi I and ligated to reconstruct a pGADT7-Sfi I three-frame vector. The ligation mixture was transformed into Escherichia coli HST08 premium electro cells by electroporation to construct the primary cDNA library. The library capacity was 1.0×10(6)clones. Gel electrophoresis results indicated that the fragments ranged from 0.5kb to 4.2kb. Randomly picked clones show that the recombination rate was 100%. The three-frame primary cDNA library plasmid mixture (5×10(5)cfu) was also transformed into E. coli HST08 premium electro cells, and all clones were harvested to amplify the cDNA library. To detect the sufficiency of the cDNA library, HAstV capsid protein as bait was screened and tested against the Caco-2 cDNA library by a yeast two-hybrid (Y2H) system. A total of 20 proteins were found to interact with the capsid protein. These results showed that a high-quality three-frame cDNA library from Caco-2 cells was successfully constructed. This library was efficient for the application to the Y2H system, and could be used for future research. Copyright © 2014 Elsevier B.V. All rights reserved.

Inferring Domain-Domain Interactions from Protein-Protein Interactions with Formal Concept Analysis

PubMed Central

Khor, Susan

2014-01-01

Identifying reliable domain-domain interactions will increase our ability to predict novel protein-protein interactions, to unravel interactions in protein complexes, and thus gain more information about the function and behavior of genes. One of the challenges of identifying reliable domain-domain interactions is domain promiscuity. Promiscuous domains are domains that can occur in many domain architectures and are therefore found in many proteins. This becomes a problem for a method where the score of a domain-pair is the ratio between observed and expected frequencies because the protein-protein interaction network is sparse. As such, many protein-pairs will be non-interacting and domain-pairs with promiscuous domains will be penalized. This domain promiscuity challenge to the problem of inferring reliable domain-domain interactions from protein-protein interactions has been recognized, and a number of work-arounds have been proposed. This paper reports on an application of Formal Concept Analysis to this problem. It is found that the relationship between formal concepts provides a natural way for rare domains to elevate the rank of promiscuous domain-pairs and enrich highly ranked domain-pairs with reliable domain-domain interactions. This piggybacking of promiscuous domain-pairs onto less promiscuous domain-pairs is possible only with concept lattices whose attribute-labels are not reduced and is enhanced by the presence of proteins that comprise both promiscuous and rare domains. PMID:24586450

A lanthipeptide library used to identify a protein-protein interaction inhibitor.

PubMed

Yang, Xiao; Lennard, Katherine R; He, Chang; Walker, Mark C; Ball, Andrew T; Doigneaux, Cyrielle; Tavassoli, Ali; van der Donk, Wilfred A

2018-04-01

In this article we describe the production and screening of a genetically encoded library of 10 6 lanthipeptides in Escherichia coli using the substrate-tolerant lanthipeptide synthetase ProcM. This plasmid-encoded library was combined with a bacterial reverse two-hybrid system for the interaction of the HIV p6 protein with the UEV domain of the human TSG101 protein, which is a critical protein-protein interaction for HIV budding from infected cells. Using this approach, we identified an inhibitor of this interaction from the lanthipeptide library, whose activity was verified in vitro and in cell-based virus-like particle-budding assays. Given the variety of lanthipeptide backbone scaffolds that may be produced with ProcM, this method may be used for the generation of genetically encoded libraries of natural product-like lanthipeptides containing substantial structural diversity. Such libraries may be combined with any cell-based assay to identify lanthipeptides with new biological activities.

Novel Hedgehog pathway targets against basal cell carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tang, Jean Y.; Department of Epidemiology and Biostatistics, University of California, San Francisco, CA; So, P.-L.

2007-11-01

The Hedgehog signaling pathway plays a key role in directing growth and patterning during embryonic development and is required in vertebrates for the normal development of many structures, including the neural tube, axial skeleton, skin, and hair. Aberrant activation of the Hedgehog (Hh) pathway in adult tissue is associated with the development of basal cell carcinoma (BCC), medulloblastoma, and a subset of pancreatic, gastrointestinal, and other cancers. This review will provide an overview of what is known about the mechanisms by which activation of Hedgehog signaling leads to the development of BCCs and will review two recent papers suggesting thatmore » agents that modulate sterol levels might influence the Hh pathway. Thus, sterols may be a new therapeutic target for the treatment of BCCs, and readily available agents such as statins (HMG-CoA reductase inhibitors) or vitamin D might be helpful in reducing BCC incidence.« less

Unilateral proptosis and orbital cellulitis in eight African hedgehogs (Atelerix albiventris).

PubMed

Wheler, C L; Grahn, B H; Pocknell, A M

2001-06-01

Eight African hedgehogs (Atelerix albiventris) were presented with unilateral proptosis. Six animals presented specifically for an ocular problem, whereas two had concurrent neurologic disease. Enucleation and light microscopic examination of tissues was performed in five animals, and euthanasia followed by complete postmortem examination was performed in three animals. Histopathologic findings in all hedgehogs included orbital cellulitis, panophthalmitis, and corneal ulceration, with perforation in seven of eight eyes. The etiology of the orbital cellulitis was not determined, but it appeared to precede proptosis. Orbits in hedgehogs are shallow and the palpebral fissures are large, which may predispose them to proptosis, similar to brachycephalic dogs. This clinical presentation was seen in 15% (8/54) of African hedgehogs presented to the Western College of Veterinary Medicine over a 2-yr period from January 1995 to December 1996 and warrants further investigation.

PREFACE: Protein protein interactions: principles and predictions

NASA Astrophysics Data System (ADS)

Nussinov, Ruth; Tsai, Chung-Jung

2005-06-01

Proteins are the `workhorses' of the cell. Their roles span functions as diverse as being molecular machines and signalling. They carry out catalytic reactions, transport, form viral capsids, traverse membranes and form regulated channels, transmit information from DNA to RNA, making possible the synthesis of new proteins, and they are responsible for the degradation of unnecessary proteins and nucleic acids. They are the vehicles of the immune response and are responsible for viral entry into the cell. Given their importance, considerable effort has been centered on the prediction of protein function. A prime way to do this is through identification of binding partners. If the function of at least one of the components with which the protein interacts is known, that should let us assign its function(s) and the pathway(s) in which it plays a role. This holds since the vast majority of their chores in the living cell involve protein-protein interactions. Hence, through the intricate network of these interactions we can map cellular pathways, their interconnectivities and their dynamic regulation. Their identification is at the heart of functional genomics; their prediction is crucial for drug discovery. Knowledge of the pathway, its topology, length, and dynamics may provide useful information for forecasting side effects. The goal of predicting protein-protein interactions is daunting. Some associations are obligatory, others are continuously forming and dissociating. In principle, from the physical standpoint, any two proteins can interact, but under what conditions and at which strength? The principles of protein-protein interactions are general: the non-covalent interactions of two proteins are largely the outcome of the hydrophobic effect, which drives the interactions. In addition, hydrogen bonds and electrostatic interactions play important roles. Thus, many of the interactions observed in vitro are the outcome of experimental overexpression. Protein disorder

Targeting Breast Cancer Recurrence via Hedgehog-mediated Sensitization of Breast Cancer Stem Cells

DTIC Science & Technology

2011-07-01

Hedgehog -mediated Sensitization of Breast Cancer Stem Cells PRINCIPAL INVESTIGATOR: David J. Robbins, Ph.D...June 2010 – 14 June 2011 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Targeting Breast Cancer Recurrence via Hedgehog -mediated Sensitization of...this award. Introduction The purpose of the research supported by this award is to determine if targeting the hedgehog signaling pathway in

Anaplastic astrocytoma in the spinal cord of an African pygmy hedgehog (Atelerix albiventris).

PubMed

Gibson, C J; Parry, N M A; Jakowski, R M; Eshar, D

2008-11-01

A 2-year-old, female hedgehog presented with an 8-month history of progressive, ascending paresis/paralysis and was tentatively diagnosed with wobbly hedgehog syndrome. She died awaiting further diagnostic tests, and the owners consented to postmortem examination. Grossly, the bladder was large and flaccid and the cervical and lumbar spinal cord were regionally enlarged, light grey, and friable with multifocal hemorrhages. The thoracic spinal cord was grossly normal. Microscopically all regions of the spinal cord had similar changes, although the cervical and lumbar sections were most severely affected. These regions were completely effaced by a moderately cellular infiltration of highly pleomorphic polygonal to spindle shaped cells, mineralization, and necrosis, which were most consistent with anaplastic astrocytoma. The thoracic spinal cord white matter was similarly infiltrated by the neoplastic cells, with perivascular extension into the otherwise normal grey matter. A diagnosis of anaplastic astrocytoma was confirmed using immunohistochemical stains that were positive for glial fibrillary acidic protein and S100.

Hedgehog signaling in the stomach.

PubMed

Konstantinou, Daniel; Bertaux-Skeirik, Nina; Zavros, Yana

2016-12-01

The Hedgehog (Hh) signaling pathway not only plays a key part in controlling embryonic development, but in the adult stomach governs important cellular events such as epithelial cell differentiation, proliferation, gastric disease, and regeneration. In particular, Sonic Hedgehog (Shh) signaling has been well studied for its role in gastric physiology and pathophysiology. Shh is secreted from the gastric parietal cells and contributes to the regeneration of the epithelium in response to injury, or the development of gastritis during Helicobacter pylori infection. Dysregulation of the Shh signaling pathway leads to the disruption of gastric differentiation, loss of gastric acid secretion and the development of cancer. In this chapter, we will review the most recent findings that reveal the role of Shh as a regulator of gastric physiology, regeneration, and disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

Interactions of proteins in human plasma with modified polystyrene resins.

PubMed

Boisson-Vidal, C; Jozefonvicz, J; Brash, J L

1991-01-01

Investigations are reported on the composition of protein layers adsorbed from plasma to various modified polystyrene resins. As well as polystyrene itself, polystyrene bearing sulfonate groups in the benzene rings, and polystyrene sulfonate in which the sulfonate groups were converted to amino acid sulfamide, were investigated. Some of these resins were shown in previous work to have anticoagulant properties. To study the adsorption of proteins from plasma, the resins were exposed to citrate anticoagulated human plasma for 3 h. Adsorbed proteins were then eluted sequentially by 1M Tris buffer and 4% SDS solution, and examined by SDS-PAGE. The gel patterns were similar on all resins except polystyrene. From the MWs of the gel bands, the major protein component appeared to be fibrinogen. Smaller amounts of plasminogen, transferrin, albumin, and IgG were also present. In addition, Ouchterlony immunoassay of the eluates from one resin gave positive identification of complement C3, fibronectin, IgG, and IgM. Many other minor gel bands remain unidentified. A consistent finding for all resins was the presence of plasmin-type fibrinogen degradation products though the amounts varied with resin type. It is concluded from this (and from experiments showing FDP formation when fibrinogen was absorbed to the resins, from buffer containing a trace of plasminogen) that the functional groups in these materials promote the adsorption of plasminogen and its activation to a plasmin-like molecule. It appears from the substantial quantities of fibrinogen adsorbed to these materials after 3 h exposure to plasma that the Vroman effect (giving transient adsorption of fibrinogen) is not operative on these materials. It is hypothesized that specific interactions occur between fibrinogen and sulfonate groups.

Reverse Nearest Neighbor Search on a Protein-Protein Interaction Network to Infer Protein-Disease Associations.

PubMed

Suratanee, Apichat; Plaimas, Kitiporn

2017-01-01

The associations between proteins and diseases are crucial information for investigating pathological mechanisms. However, the number of known and reliable protein-disease associations is quite small. In this study, an analysis framework to infer associations between proteins and diseases was developed based on a large data set of a human protein-protein interaction network integrating an effective network search, namely, the reverse k -nearest neighbor (R k NN) search. The R k NN search was used to identify an impact of a protein on other proteins. Then, associations between proteins and diseases were inferred statistically. The method using the R k NN search yielded a much higher precision than a random selection, standard nearest neighbor search, or when applying the method to a random protein-protein interaction network. All protein-disease pair candidates were verified by a literature search. Supporting evidence for 596 pairs was identified. In addition, cluster analysis of these candidates revealed 10 promising groups of diseases to be further investigated experimentally. This method can be used to identify novel associations to better understand complex relationships between proteins and diseases.

Characterization of the human oncogene SCL/TAL1 interrupting locus (Stil) mediated Sonic hedgehog (Shh) signaling transduction in proliferating mammalian dopaminergic neurons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Lei; Department of Physiology, Nankai University School of Medicine, Tianjin 300071; Carr, Aprell L.

2014-07-11

Highlights: • Stil is a human oncogene that is conserved in vertebrate species. • Stil functions in the Shh pathway in mammalian cells. • The expression of Stil is required for mammalian dopaminergic cell proliferation. - Abstract: The human oncogene SCL/TAL1 interrupting locus (Stil) is highly conserved in all vertebrate species. In humans, the expression of Stil is involved in cancer cell survival, apoptosis and proliferation. In this research, we investigated the roles of Stil expression in cell proliferation of mammalian dopaminergic (DA) PC12 cells. Stil functions through the Sonic hedgehog (Shh) signal transduction pathway. Co-immunoprecipitation tests revealed that STILmore » interacts with Shh downstream components, which include SUFU and GLI1. By examining the expression of Stil, Gli1, CyclinD2 (cell-cycle marker) and PCNA (proliferating cell nuclear antigen), we found that up-regulation of Stil expression (transfection with overexpression plasmids) increased Shh signaling transduction and PC12 cell proliferation, whereas down-regulation of Stil expression (by shRNA) inhibited Shh signaling transduction, and thereby decreased PC12 cell proliferation. Transient transfection of PC12 cells with Stil knockdown or overexpression plasmids did not affect PC12 cell neural differentiation, further indicating the specific roles of Stil in cell proliferation. The results from this research suggest that Stil may serve as a bio-marker for neurological diseases involved in DA neurons, such as Parkinson’s disease.« less

Detecting protein-protein interactions using Renilla luciferase fusion proteins.

PubMed

Burbelo, Peter D; Kisailus, Adam E; Peck, Jeremy W

2002-11-01

We have developed a novel system designated the luciferase assay for protein detection (LAPD) to study protein-protein interactions. This method involves two protein fusions, a soluble reporter fusion and a fusion for immobilizing the target protein. The soluble reporter is an N-terminal Renilla luciferase fusion protein that exhibits high Renilla luciferase activity. Crude cleared lysates from transfected Cos1 cells that express the Renilla luciferase fusion protein can be used in binding assays with immobilized target proteins. Following incubation and washing, target-bound Renilla luciferase fusion proteins produce light from the coelenterazine substrate, indicating an interaction between the two proteins of interest. As proof of the principle, we reproduced known, transient protein-protein interactions between the Cdc42 GTPase and its effector proteins. GTPase Renilla fusion proteins produced in Cos1 cells were tested with immobilized recombinant GST-N-WASP and CEP5 effector proteins. Using this assay, we could detect specific interactions of Cdc42 with these effector proteins in approximately 50 min. The specificity of these interactions was demonstrated by showing that they were GTPase-specific and GTP-dependent and not seen with other unrelated target proteins. These results suggest that the LAPD method, which is both rapid and sensitive, may have research and practical applications.

Spontaneous tumours in captive African hedgehogs (Atelerix albiventris): a retrospective study.

PubMed

Raymond, J T; Garner, M M

2001-01-01

Forty tumours were diagnosed in 35 (53%) of 66 captive African hedgehogs documented at Northwest ZooPath (NZP) between 1994 and 1999. Three hedgehogs had more than one type of tumour and the remaining 32 had a single type. Of the 35 hedgehogs with tumours, 14 were female, 11 were male, and 10 were of unknown gender; 21 were from zoological parks and 14 were privately owned. Twenty of the hedgehogs with tumours were adult (>1 year old) with a median age of 3.5 years (range 2-5.5 years); 15, of unreported age, were classified as adult. Thirty-four (85%) of the 40 tumours were classified as malignant and six (15%) as benign. The integumentary, haemolymphatic, digestive and endocrine systems were common sites for tumours. The most common tumours were mammary gland adenocarcinoma, lympho-sarcoma and oral squamous cell carcinoma. Copyright Harcourt Publishers Ltd.

Predicting PDZ domain mediated protein interactions from structure

PubMed Central

2013-01-01

Background PDZ domains are structural protein domains that recognize simple linear amino acid motifs, often at protein C-termini, and mediate protein-protein interactions (PPIs) in important biological processes, such as ion channel regulation, cell polarity and neural development. PDZ domain-peptide interaction predictors have been developed based on domain and peptide sequence information. Since domain structure is known to influence binding specificity, we hypothesized that structural information could be used to predict new interactions compared to sequence-based predictors. Results We developed a novel computational predictor of PDZ domain and C-terminal peptide interactions using a support vector machine trained with PDZ domain structure and peptide sequence information. Performance was estimated using extensive cross validation testing. We used the structure-based predictor to scan the human proteome for ligands of 218 PDZ domains and show that the predictions correspond to known PDZ domain-peptide interactions and PPIs in curated databases. The structure-based predictor is complementary to the sequence-based predictor, finding unique known and novel PPIs, and is less dependent on training–testing domain sequence similarity. We used a functional enrichment analysis of our hits to create a predicted map of PDZ domain biology. This map highlights PDZ domain involvement in diverse biological processes, some only found by the structure-based predictor. Based on this analysis, we predict novel PDZ domain involvement in xenobiotic metabolism and suggest new interactions for other processes including wound healing and Wnt signalling. Conclusions We built a structure-based predictor of PDZ domain-peptide interactions, which can be used to scan C-terminal proteomes for PDZ interactions. We also show that the structure-based predictor finds many known PDZ mediated PPIs in human that were not found by our previous sequence-based predictor and is less dependent on

Hedgehog signaling regulates gene expression in planarian glia.

PubMed

Wang, Irving E; Lapan, Sylvain W; Scimone, M Lucila; Clandinin, Thomas R; Reddien, Peter W

2016-09-09

Hedgehog signaling is critical for vertebrate central nervous system (CNS) development, but its role in CNS biology in other organisms is poorly characterized. In the planarian Schmidtea mediterranea, hedgehog (hh ) is expressed in medial cephalic ganglia neurons, suggesting a possible role in CNS maintenance or regeneration. We performed RNA sequencing of planarian brain tissue following RNAi of hh and patched (ptc) , which encodes the Hh receptor. Two misregulated genes, intermediate filament-1 (if-1 ) and calamari (cali ), were expressed in a previously unidentified non-neural CNS cell type. These cells expressed orthologs of astrocyte-associated genes involved in neurotransmitter uptake and metabolism, and extended processes enveloping regions of high synapse concentration. We propose that these cells are planarian glia. Planarian glia were distributed broadly, but only expressed if-1 and cali in the neuropil near hh + neurons. Planarian glia and their regulation by Hedgehog signaling present a novel tractable system for dissection of glia biology.

Interaction of gold and silver nanoparticles with human plasma: Analysis of protein corona reveals specific binding patterns.

PubMed

Lai, Wenjia; Wang, Qingsong; Li, Lumeng; Hu, Zhiyuan; Chen, Jiankui; Fang, Qiaojun

2017-04-01

Determining how nanomaterials interact with plasma will assist in understanding their effects on the biological system. This work presents a systematic study of the protein corona formed from human plasma on 20nm silver and gold nanoparticles with three different surface modifications, including positive and negative surface charges. The results show that all nanoparticles, even those with positive surface modifications, acquire negative charges after interacting with plasma. Approximately 300 proteins are identified on the coronas, while 99 are commonly found on each nanomaterial. The 20 most abundant proteins account for over 80% of the total proteins abundance. Remarkably, the surface charge and core of the nanoparticles, as well as the isoelectric point of the plasma proteins, are found to play significant roles in determining the nanoparticle coronas. Albumin and globulins are present at levels of less than 2% on these nanoparticle coronas. Fibrinogen, which presents in the plasma but not in the serum, preferably binds to negatively charged gold nanoparticles. These observations demonstrate the specific plasma protein binding pattern of silver and gold nanoparticles, as well as the importance of the surface charge and core in determining the protein corona compositions. The potential downstream biological impacts of the corona proteins were also investigated. Copyright © 2017 Elsevier B.V. All rights reserved.

Human I-mfa domain proteins specifically interact with KSHV LANA and affect its regulation of Wnt signaling-dependent transcription

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kusano, Shuichi, E-mail: skusano@m2.kufm.kagoshima-u.ac.jp; Eizuru, Yoshito

2010-06-04

Kaposi's sarcoma-associated herpes virus (KSHV)-encoded latency-associated nuclear antigen (LANA) protein has been reported to interact with glycogen synthase kinase 3{beta} (GSK-3{beta}) and to negatively regulate its activity, leading to stimulation of GSK-3{beta}-dependent {beta}-catenin degradation. We show here that the I-mfa domain proteins, HIC (human I-mfa domain-containing protein) and I-mfa (inhibitor of MyoD family a), interacted in vivo with LANA through their C-terminal I-mfa domains. This interaction affected the intracellular localization of HIC, inhibited the LANA-dependent transactivation of a {beta}-catenin-regulated reporter construct, and decreased the level of the LANA.GSK-3{beta} complex. These data reveal for the first time that I-mfa domain proteinsmore » interact with LANA and negatively regulate LANA-mediated activation of Wnt signaling-dependent transcription by inhibiting the formation of the LANA.GSK-3{beta} complex.« less

Evolutionary Genomics and Adaptive Evolution of the Hedgehog Gene Family (Shh, Ihh and Dhh) in Vertebrates

PubMed Central

Pereira, Joana; Johnson, Warren E.; O’Brien, Stephen J.; Jarvis, Erich D.; Zhang, Guojie; Gilbert, M. Thomas P.; Vasconcelos, Vitor; Antunes, Agostinho

2014-01-01

The Hedgehog (Hh) gene family codes for a class of secreted proteins composed of two active domains that act as signalling molecules during embryo development, namely for the development of the nervous and skeletal systems and the formation of the testis cord. While only one Hh gene is found typically in invertebrate genomes, most vertebrates species have three (Sonic hedgehog – Shh; Indian hedgehog – Ihh; and Desert hedgehog – Dhh), each with different expression patterns and functions, which likely helped promote the increasing complexity of vertebrates and their successful diversification. In this study, we used comparative genomic and adaptive evolutionary analyses to characterize the evolution of the Hh genes in vertebrates following the two major whole genome duplication (WGD) events. To overcome the lack of Hh-coding sequences on avian publicly available databases, we used an extensive dataset of 45 avian and three non-avian reptilian genomes to show that birds have all three Hh paralogs. We find suggestions that following the WGD events, vertebrate Hh paralogous genes evolved independently within similar linkage groups and under different evolutionary rates, especially within the catalytic domain. The structural regions around the ion-binding site were identified to be under positive selection in the signaling domain. These findings contrast with those observed in invertebrates, where different lineages that experienced gene duplication retained similar selective constraints in the Hh orthologs. Our results provide new insights on the evolutionary history of the Hh gene family, the functional roles of these paralogs in vertebrate species, and on the location of mutational hotspots. PMID:25549322

Hedgehog-GLI signaling drives self-renewal and tumorigenicity of human melanoma-initiating cells.

PubMed

Santini, Roberta; Vinci, Maria C; Pandolfi, Silvia; Penachioni, Junia Y; Montagnani, Valentina; Olivito, Biagio; Gattai, Riccardo; Pimpinelli, Nicola; Gerlini, Gianni; Borgognoni, Lorenzo; Stecca, Barbara

2012-09-01

The question of whether cancer stem/tumor-initiating cells (CSC/TIC) exist in human melanomas has arisen in the last few years. Here, we have used nonadherent spheres and the aldehyde dehydrogenase (ALDH) enzymatic activity to enrich for CSC/TIC in a collection of human melanomas obtained from a broad spectrum of sites and stages. We find that melanomaspheres display extensive in vitro self-renewal ability and sustain tumor growth in vivo, generating human melanoma xenografts that recapitulate the phenotypic composition of the parental tumor. Melanomaspheres express high levels of Hedgehog (HH) pathway components and of embryonic pluripotent stem cell factors SOX2, NANOG, OCT4, and KLF4. We show that human melanomas contain a subset of cells expressing high ALDH activity (ALDH(high)), which is endowed with higher self-renewal and tumorigenic abilities than the ALDH(low) population. A good correlation between the number of ALDH(high) cells and sphere formation efficiency was observed. Notably, both pharmacological inhibition of HH signaling by the SMOOTHENED (SMO) antagonist cyclopamine and GLI antagonist GANT61 and stable expression of shRNA targeting either SMO or GLI1 result in a significant decrease in melanoma stem cell self-renewal in vitro and a reduction in the number of ALDH(high) melanoma stem cells. Finally, we show that interference with the HH-GLI pathway through lentiviral-mediated silencing of SMO and GLI1 drastically diminishes tumor initiation of ALDH(high) melanoma stem cells. In conclusion, our data indicate an essential role of the HH-GLI1 signaling in controlling self-renewal and tumor initiation of melanoma CSC/TIC. Targeting HH-GLI1 is thus predicted to reduce the melanoma stem cell compartment. Copyright © 2012 AlphaMed Press.

Controllability of protein-protein interaction phosphorylation-based networks: Participation of the hub 14-3-3 protein family

PubMed Central

Uhart, Marina; Flores, Gabriel; Bustos, Diego M.

2016-01-01

Posttranslational regulation of protein function is an ubiquitous mechanism in eukaryotic cells. Here, we analyzed biological properties of nodes and edges of a human protein-protein interaction phosphorylation-based network, especially of those nodes critical for the network controllability. We found that the minimal number of critical nodes needed to control the whole network is 29%, which is considerably lower compared to other real networks. These critical nodes are more regulated by posttranslational modifications and contain more binding domains to these modifications than other kinds of nodes in the network, suggesting an intra-group fast regulation. Also, when we analyzed the edges characteristics that connect critical and non-critical nodes, we found that the former are enriched in domain-to-eukaryotic linear motif interactions, whereas the later are enriched in domain-domain interactions. Our findings suggest a possible structure for protein-protein interaction networks with a densely interconnected and self-regulated central core, composed of critical nodes with a high participation in the controllability of the full network, and less regulated peripheral nodes. Our study offers a deeper understanding of complex network control and bridges the controllability theorems for complex networks and biological protein-protein interaction phosphorylation-based networked systems. PMID:27195976

Radiological investigations of the hedgehog (Erinaceus concolor) appendicular skeleton.

PubMed

Hashemi, Mohammad; Javadi, Shahram; Hadian, Mojtaba; Pourreza, Behzad; Behfar, Mahdi

2009-03-01

The normal radiographic anatomy of the healthy hedgehog can help to identify anatomic features unique to the hedgehog while comparing it with other small mammals, such as the dog and cat. Radiographic examination is a method that can play an important role in the diagnosis of a wide variety of skeletal diseases. Seven (2 males, 5 females) free-living hedgehogs (Erinaceus concolor) from the Urmia region of Iran were selected for this study. Lateral and craniocaudal radiographs from the front and hind limbs were obtained. The radiographs from these hedgehogs were compared with the normal canine and feline skeletal radiographic anatomy. On the forelimb radiographs, the clavicle was observed as a complete bone connected to the scapula and manubrium. There are three and five carpal bones in the proximal and distal rows, respectively, as in the dog and cat. The pelvis has a larger obturator foramen when compared with the dog and cat. In the lateral view, the pubis and ischium are relatively larger than in the dog and cat and have a more ventral position. The tarsal bones are similar to those of the dog and cat. The number of phalanges and sesamoid bones in the forelimb and hindlimb are likewise similar to those found in the dog and cat.

Effects of a natural fire on a Kuenzler's hedgehog cactus (Echinocereus fendleri var. kuenzleri) and nylon hedgehog cactus (Echinocereus viridiflorus) population in Southeastern New Mexico

Treesearch

Robert C. Sivinski

2007-01-01

During the summer of 1992, a natural wildfire burned 250 acres of juniper savanna on Rawhide Ridge in the Guadalupe Mountains of southeastern New Mexico. This fire burned through the center of a Kuenzler's hedgehog cactus population. This threatened cactus is locally sympatric with the more abundant nylon hedgehog cactus, which has similar growth form and stature...

Interaction entropy for protein-protein binding.

PubMed

Sun, Zhaoxi; Yan, Yu N; Yang, Maoyou; Zhang, John Z H

2017-03-28

Protein-protein interactions are at the heart of signal transduction and are central to the function of protein machine in biology. The highly specific protein-protein binding is quantitatively characterized by the binding free energy whose accurate calculation from the first principle is a grand challenge in computational biology. In this paper, we show how the interactionentropy approach, which was recently proposed for protein-ligand binding free energy calculation, can be applied to computing the entropic contribution to the protein-protein binding free energy. Explicit theoretical derivation of the interactionentropy approach for protein-protein interaction system is given in detail from the basic definition. Extensive computational studies for a dozen realistic protein-protein interaction systems are carried out using the present approach and comparisons of the results for these protein-protein systems with those from the standard normal mode method are presented. Analysis of the present method for application in protein-protein binding as well as the limitation of the method in numerical computation is discussed. Our study and analysis of the results provided useful information for extracting correct entropic contribution in protein-protein binding from molecular dynamics simulations.

Enhancing interacting residue prediction with integrated contact matrix prediction in protein-protein interaction.

PubMed

Du, Tianchuan; Liao, Li; Wu, Cathy H

2016-12-01

Identifying the residues in a protein that are involved in protein-protein interaction and identifying the contact matrix for a pair of interacting proteins are two computational tasks at different levels of an in-depth analysis of protein-protein interaction. Various methods for solving these two problems have been reported in the literature. However, the interacting residue prediction and contact matrix prediction were handled by and large independently in those existing methods, though intuitively good prediction of interacting residues will help with predicting the contact matrix. In this work, we developed a novel protein interacting residue prediction system, contact matrix-interaction profile hidden Markov model (CM-ipHMM), with the integration of contact matrix prediction and the ipHMM interaction residue prediction. We propose to leverage what is learned from the contact matrix prediction and utilize the predicted contact matrix as "feedback" to enhance the interaction residue prediction. The CM-ipHMM model showed significant improvement over the previous method that uses the ipHMM for predicting interaction residues only. It indicates that the downstream contact matrix prediction could help the interaction site prediction.

Computational prediction of protein interactions related to the invasion of erythrocytes by malarial parasites.

PubMed

Liu, Xuewu; Huang, Yuxiao; Liang, Jiao; Zhang, Shuai; Li, Yinghui; Wang, Jun; Shen, Yan; Xu, Zhikai; Zhao, Ya

2014-11-30

The invasion of red blood cells (RBCs) by malarial parasites is an essential step in the life cycle of Plasmodium falciparum. Human-parasite surface protein interactions play a critical role in this process. Although several interactions between human and parasite proteins have been discovered, the mechanism related to invasion remains poorly understood because numerous human-parasite protein interactions have not yet been identified. High-throughput screening experiments are not feasible for malarial parasites due to difficulty in expressing the parasite proteins. Here, we performed computational prediction of the PPIs involved in malaria parasite invasion to elucidate the mechanism by which invasion occurs. In this study, an expectation maximization algorithm was used to estimate the probabilities of domain-domain interactions (DDIs). Estimates of DDI probabilities were then used to infer PPI probabilities. We found that our prediction performance was better than that based on the information of D. melanogaster alone when information related to the six species was used. Prediction performance was assessed using protein interaction data from S. cerevisiae, indicating that the predicted results were reliable. We then used the estimates of DDI probabilities to infer interactions between 490 parasite and 3,787 human membrane proteins. A small-scale dataset was used to illustrate the usability of our method in predicting interactions between human and parasite proteins. The positive predictive value (PPV) was lower than that observed in S. cerevisiae. We integrated gene expression data to improve prediction accuracy and to reduce false positives. We identified 80 membrane proteins highly expressed in the schizont stage by fast Fourier transform method. Approximately 221 erythrocyte membrane proteins were identified using published mass spectral datasets. A network consisting of 205 interactions was predicted. Results of network analysis suggest that SNARE proteins of

Using the clustered circular layout as an informative method for visualizing protein-protein interaction networks.

PubMed

Fung, David C Y; Wilkins, Marc R; Hart, David; Hong, Seok-Hee

2010-07-01

The force-directed layout is commonly used in computer-generated visualizations of protein-protein interaction networks. While it is good for providing a visual outline of the protein complexes and their interactions, it has two limitations when used as a visual analysis method. The first is poor reproducibility. Repeated running of the algorithm does not necessarily generate the same layout, therefore, demanding cognitive readaptation on the investigator's part. The second limitation is that it does not explicitly display complementary biological information, e.g. Gene Ontology, other than the protein names or gene symbols. Here, we present an alternative layout called the clustered circular layout. Using the human DNA replication protein-protein interaction network as a case study, we compared the two network layouts for their merits and limitations in supporting visual analysis.

Hedgehog-mediated mesenchymal-epithelial interactions modulate hepatic response to bile duct ligation.

PubMed

Omenetti, Alessia; Yang, Liu; Li, Yin-Xiong; McCall, Shannon J; Jung, Youngmi; Sicklick, Jason K; Huang, Jiawen; Choi, Steve; Suzuki, Ayako; Diehl, Anna Mae

2007-05-01

In bile duct-ligated (BDL) rodents, as in humans with chronic cholangiopathies, biliary obstruction triggers proliferation of bile ductular cells that are surrounded by fibrosis produced by adjacent myofibroblastic cells in the hepatic mesenchyme. The proximity of the myofibroblasts and cholangiocytes suggests that mesenchymal-epithelial crosstalk promotes the fibroproliferative response to cholestatic liver injury. Studying BDL mice, we found that bile duct obstruction induces activity of the Hedgehog (Hh) pathway, a system that regulates the viability and differentiation of various progenitors during embryogenesis. After BDL, many bile ductular cells and fibroblastic-appearing cells in the portal stroma express Hh ligands, receptor and/or target genes. Transwell cocultures of an immature cholangiocyte line that expresses the Hh receptor, Patched (Ptc), with liver myofibroblastic cells demonstrated that both cell types produced Hh ligands that enhanced each other's viability and proliferation. Further support for the concept that Hh signaling modulates the response to BDL was generated by studying PtcLacZ mice, which have an impaired ability to constrain Hh signaling due to a heterozygous deficiency of Ptc. After BDL, PtcLacZ mice upregulated fibrosis gene expression earlier than wild-type controls and manifested an unusually intense ductular reaction, more expanded fibrotic portal areas, and a greater number of lobular necrotic foci. Our findings reveal that adult livers resurrect developmental signaling systems, such as the Hh pathway, to guide remodeling of the biliary epithelia and stroma after cholestatic injury.

Glypican-5 stimulates rhabdomyosarcoma cell proliferation by activating Hedgehog signaling

PubMed Central

Shi, Wen; Capurro, Mariana

2011-01-01

Glypican-5 (GPC5) is one of the six members of the glypican family. It has been previously reported that GPC5 stimulates the proliferation of rhabdomyosarcoma cells. In this study, we show that this stimulatory activity of GPC5 is a result of its ability to promote Hedgehog (Hh) signaling. We have previously shown that GPC3, another member of the glypican family, inhibits Hh signaling by competing with Patched 1 (Ptc1) for Hh binding. Furthermore, we showed that GPC3 binds to Hh through its core protein but not to Ptc1. In this paper, we demonstrate that GPC5 increases the binding of Sonic Hh to Ptc1. We also show that GPC5 binds to both Hh and Ptc1 through its glycosaminoglycan chains and that, unlike GPC3, GPC5 localizes to the primary cilia. Interestingly, we found that the heparan sulfate chains of GPC5 display a significantly higher degree of sulfation than those of GPC3. Based on these results, we propose that GPC5 stimulates Hh signaling by facilitating/stabilizing the interaction between Hh and Ptc1. PMID:21339334

The Kto-Skd complex can regulate ptc expression by interacting with Cubitus interruptus (Ci) in the Hedgehog signaling pathway.

PubMed

Mao, Feifei; Yang, Xiaofeng; Fu, Lin; Lv, Xiangdong; Zhang, Zhao; Wu, Wenqing; Yang, Siqi; Zhou, Zhaocai; Zhang, Lei; Zhao, Yun

2014-08-08

The hedgehog (Hh) signaling pathway plays a very important role in metazoan development by controlling pattern formation. Drosophila imaginal discs are subdivided into anterior and posterior compartments that derive from adjacent cell populations. The anterior/posterior (A/P) boundaries, which are critical to maintaining the position of organizers, are established by a complex mechanism involving Hh signaling. Here, we uncover the regulation of ptc in the Hh signaling pathway by two subunits of mediator complex, Kto and Skd, which can also regulate boundary location. Collectively, we provide further evidence that Kto-Skd affects the A/P-axial development of the whole wing disc. Kto can interact with Cubitus interruptus (Ci), bind to the Ci-binding region on ptc promoter, which are both regulated by Hh signals to down-regulate ptc expression. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor

PubMed Central

Seidel, Kerstin; Ahn, Christina P.; Lyons, David; Nee, Alexander; Ting, Kevin; Brownell, Isaac; Cao, Tim; Carano, Richard A. D.; Curran, Tom; Schober, Markus; Fuchs, Elaine; Joyner, Alexandra; Martin, Gail R.; de Sauvage, Frederic J.; Klein, Ophir D.

2010-01-01

In many organ systems such as the skin, gastrointestinal tract and hematopoietic system, homeostasis is dependent on the continuous generation of differentiated progeny from stem cells. The rodent incisor, unlike human teeth, grows throughout the life of the animal and provides a prime example of an organ that rapidly deteriorates if newly differentiated cells cease to form from adult stem cells. Hedgehog (Hh) signaling has been proposed to regulate self-renewal, survival, proliferation and/or differentiation of stem cells in several systems, but to date there is little evidence supporting a role for Hh signaling in adult stem cells. We used in vivo genetic lineage tracing to identify Hh-responsive stem cells in the mouse incisor and we show that sonic hedgehog (SHH), which is produced by the differentiating progeny of the stem cells, signals to several regions of the incisor. Using a hedgehog pathway inhibitor (HPI), we demonstrate that Hh signaling is not required for stem cell survival but is essential for the generation of ameloblasts, one of the major differentiated cell types in the tooth, from the stem cells. These results therefore reveal the existence of a positive-feedback loop in which differentiating progeny produce the signal that in turn allows them to be generated from stem cells. PMID:20978073

Protein-Protein Interaction Site Predictions with Three-Dimensional Probability Distributions of Interacting Atoms on Protein Surfaces

PubMed Central

Chen, Ching-Tai; Peng, Hung-Pin; Jian, Jhih-Wei; Tsai, Keng-Chang; Chang, Jeng-Yih; Yang, Ei-Wen; Chen, Jun-Bo; Ho, Shinn-Ying; Hsu, Wen-Lian; Yang, An-Suei

2012-01-01

Protein-protein interactions are key to many biological processes. Computational methodologies devised to predict protein-protein interaction (PPI) sites on protein surfaces are important tools in providing insights into the biological functions of proteins and in developing therapeutics targeting the protein-protein interaction sites. One of the general features of PPI sites is that the core regions from the two interacting protein surfaces are complementary to each other, similar to the interior of proteins in packing density and in the physicochemical nature of the amino acid composition. In this work, we simulated the physicochemical complementarities by constructing three-dimensional probability density maps of non-covalent interacting atoms on the protein surfaces. The interacting probabilities were derived from the interior of known structures. Machine learning algorithms were applied to learn the characteristic patterns of the probability density maps specific to the PPI sites. The trained predictors for PPI sites were cross-validated with the training cases (consisting of 432 proteins) and were tested on an independent dataset (consisting of 142 proteins). The residue-based Matthews correlation coefficient for the independent test set was 0.423; the accuracy, precision, sensitivity, specificity were 0.753, 0.519, 0.677, and 0.779 respectively. The benchmark results indicate that the optimized machine learning models are among the best predictors in identifying PPI sites on protein surfaces. In particular, the PPI site prediction accuracy increases with increasing size of the PPI site and with increasing hydrophobicity in amino acid composition of the PPI interface; the core interface regions are more likely to be recognized with high prediction confidence. The results indicate that the physicochemical complementarity patterns on protein surfaces are important determinants in PPIs, and a substantial portion of the PPI sites can be predicted correctly with

Targeting RNA–Protein Interactions within the Human Immunodeficiency Virus Type 1 Lifecycle

PubMed Central

2013-01-01

RNA–protein interactions are vital throughout the HIV-1 life cycle for the successful production of infectious virus particles. One such essential RNA–protein interaction occurs between the full-length genomic viral RNA and the major structural protein of the virus. The initial interaction is between the Gag polyprotein and the viral RNA packaging signal (psi or Ψ), a highly conserved RNA structural element within the 5′-UTR of the HIV-1 genome, which has gained attention as a potential therapeutic target. Here, we report the application of a target-based assay to identify small molecules, which modulate the interaction between Gag and Ψ. We then demonstrate that one such molecule exhibits potent inhibitory activity in a viral replication assay. The mode of binding of the lead molecules to the RNA target was characterized by 1H NMR spectroscopy. PMID:24358934

Infection with Crenosoma striatum lungworm in Long-eared Hedgehog (Hemiechinus auritus) in Kerman province southeast of Iran.

PubMed

Mirzaei, Mohammad

2014-12-01

Hedgehogs are distributed in different areas of Iran. Unfortunately, clinical and parasitological studies on parasites of hedgehogs are very few. Crenosoma striatum is a common lungworm in hedgehogs. C. striatum infection can cause weight loss, dry cough, bronchitis with ulcerous reactions based on secondary bacterial infections, pulmonary damage, thickening of the tracheal wall, and pulmonary emphysema up to cardiovascular failure. In this survey, six dead hedgehogs (Hemiechinus auritus) were investigated for lungworm infection. All the six hedgehogs had C. striatum infection in their lungs.

NOXclass: prediction of protein-protein interaction types.

PubMed

Zhu, Hongbo; Domingues, Francisco S; Sommer, Ingolf; Lengauer, Thomas

2006-01-19

Structural models determined by X-ray crystallography play a central role in understanding protein-protein interactions at the molecular level. Interpretation of these models requires the distinction between non-specific crystal packing contacts and biologically relevant interactions. This has been investigated previously and classification approaches have been proposed. However, less attention has been devoted to distinguishing different types of biological interactions. These interactions are classified as obligate and non-obligate according to the effect of the complex formation on the stability of the protomers. So far no automatic classification methods for distinguishing obligate, non-obligate and crystal packing interactions have been made available. Six interface properties have been investigated on a dataset of 243 protein interactions. The six properties have been combined using a support vector machine algorithm, resulting in NOXclass, a classifier for distinguishing obligate, non-obligate and crystal packing interactions. We achieve an accuracy of 91.8% for the classification of these three types of interactions using a leave-one-out cross-validation procedure. NOXclass allows the interpretation and analysis of protein quaternary structures. In particular, it generates testable hypotheses regarding the nature of protein-protein interactions, when experimental results are not available. We expect this server will benefit the users of protein structural models, as well as protein crystallographers and NMR spectroscopists. A web server based on the method and the datasets used in this study are available at http://noxclass.bioinf.mpi-inf.mpg.de/.

Bioinformatic prediction and in vivo validation of residue-residue interactions in human proteins

NASA Astrophysics Data System (ADS)

Jordan, Daniel; Davis, Erica; Katsanis, Nicholas; Sunyaev, Shamil

2014-03-01

Identifying residue-residue interactions in protein molecules is important for understanding both protein structure and function in the context of evolutionary dynamics and medical genetics. Such interactions can be difficult to predict using existing empirical or physical potentials, especially when residues are far from each other in sequence space. Using a multiple sequence alignment of 46 diverse vertebrate species we explore the space of allowed sequences for orthologous protein families. Amino acid changes that are known to damage protein function allow us to identify specific changes that are likely to have interacting partners. We fit the parameters of the continuous-time Markov process used in the alignment to conclude that these interactions are primarily pairwise, rather than higher order. Candidates for sites under pairwise epistasis are predicted, which can then be tested by experiment. We report the results of an initial round of in vivo experiments in a zebrafish model that verify the presence of multiple pairwise interactions predicted by our model. These experimentally validated interactions are novel, distant in sequence, and are not readily explained by known biochemical or biophysical features.

Topology-function conservation in protein-protein interaction networks.

PubMed

Davis, Darren; Yaveroğlu, Ömer Nebil; Malod-Dognin, Noël; Stojmirovic, Aleksandar; Pržulj, Nataša

2015-05-15

Proteins underlay the functioning of a cell and the wiring of proteins in protein-protein interaction network (PIN) relates to their biological functions. Proteins with similar wiring in the PIN (topology around them) have been shown to have similar functions. This property has been successfully exploited for predicting protein functions. Topological similarity is also used to guide network alignment algorithms that find similarly wired proteins between PINs of different species; these similarities are used to transfer annotation across PINs, e.g. from model organisms to human. To refine these functional predictions and annotation transfers, we need to gain insight into the variability of the topology-function relationships. For example, a function may be significantly associated with specific topologies, while another function may be weakly associated with several different topologies. Also, the topology-function relationships may differ between different species. To improve our understanding of topology-function relationships and of their conservation among species, we develop a statistical framework that is built upon canonical correlation analysis. Using the graphlet degrees to represent the wiring around proteins in PINs and gene ontology (GO) annotations to describe their functions, our framework: (i) characterizes statistically significant topology-function relationships in a given species, and (ii) uncovers the functions that have conserved topology in PINs of different species, which we term topologically orthologous functions. We apply our framework to PINs of yeast and human, identifying seven biological process and two cellular component GO terms to be topologically orthologous for the two organisms. © The Author 2015. Published by Oxford University Press.

Identification of host proteins, Spata3 and Dkk2, interacting with Toxoplasma gondii micronemal protein MIC3.

PubMed

Wang, Yifan; Fang, Rui; Yuan, Yuan; Pan, Ming; Hu, Min; Zhou, Yanqin; Shen, Bang; Zhao, Junlong

2016-07-01

As an obligate intracellular protozoan, Toxoplasma gondii is a successful pathogen infecting a variety of animals, including humans. As an adhesin involving in host invasion, the micronemal protein MIC3 plays important roles in host cell attachment, as well as modulation of host EGFR signaling cascade. However, the specific host proteins that interact with MIC3 are unknown and the identification of such proteins will increase our understanding of how MIC3 exerts its functions. This study was designed to identify host proteins interacting with MIC3 by yeast two-hybrid screens. Using MIC3 as bait, a library expressing mouse proteins was screened, uncovering eight mouse proteins that showed positive interactions with MIC3. Two of which, spermatogenesis-associated protein 3 (Spata3) and dickkopf-related protein 2 (Dkk2), were further confirmed to interact with MIC3 by additional protein-protein interaction tests. The results also revealed that the tandem repeat EGF domains of MIC3 were critical in mediating the interactions with the identified host proteins. This is the first study to show that MIC3 interacts with host proteins that are involved in reproduction, growth, and development. The results will provide a clearer understanding of the functions of adhesion-associated micronemal proteins in T. gondii.

Molecular simulations of lipid-mediated protein-protein interactions.

PubMed

de Meyer, Frédérick Jean-Marie; Venturoli, Maddalena; Smit, Berend

2008-08-01

Recent experimental results revealed that lipid-mediated interactions due to hydrophobic forces may be important in determining the protein topology after insertion in the membrane, in regulating the protein activity, in protein aggregation and in signal transduction. To gain insight into the lipid-mediated interactions between two intrinsic membrane proteins, we developed a mesoscopic model of a lipid bilayer with embedded proteins, which we studied with dissipative particle dynamics. Our calculations of the potential of mean force between transmembrane proteins show that hydrophobic forces drive long-range protein-protein interactions and that the nature of these interactions depends on the length of the protein hydrophobic segment, on the three-dimensional structure of the protein and on the properties of the lipid bilayer. To understand the nature of the computed potentials of mean force, the concept of hydrophilic shielding is introduced. The observed protein interactions are interpreted as resulting from the dynamic reorganization of the system to maintain an optimal hydrophilic shielding of the protein and lipid hydrophobic parts, within the constraint of the flexibility of the components. Our results could lead to a better understanding of several membrane processes in which protein interactions are involved.

Hedgehog signaling regulates gene expression in planarian glia

PubMed Central

Wang, Irving E; Lapan, Sylvain W; Scimone, M Lucila; Clandinin, Thomas R; Reddien, Peter W

2016-01-01

Hedgehog signaling is critical for vertebrate central nervous system (CNS) development, but its role in CNS biology in other organisms is poorly characterized. In the planarian Schmidtea mediterranea, hedgehog (hh) is expressed in medial cephalic ganglia neurons, suggesting a possible role in CNS maintenance or regeneration. We performed RNA sequencing of planarian brain tissue following RNAi of hh and patched (ptc), which encodes the Hh receptor. Two misregulated genes, intermediate filament-1 (if-1) and calamari (cali), were expressed in a previously unidentified non-neural CNS cell type. These cells expressed orthologs of astrocyte-associated genes involved in neurotransmitter uptake and metabolism, and extended processes enveloping regions of high synapse concentration. We propose that these cells are planarian glia. Planarian glia were distributed broadly, but only expressed if-1 and cali in the neuropil near hh+ neurons. Planarian glia and their regulation by Hedgehog signaling present a novel tractable system for dissection of glia biology. DOI: http://dx.doi.org/10.7554/eLife.16996.001 PMID:27612382

Holoprosencephaly: from Homer to Hedgehog.

PubMed

Ming, J E; Muenke, M

1998-03-01

Holoprosencephaly (HPE), a common developmental defect affecting the forebrain and face, is etiologically heterogeneous and exhibits wide phenotypic variation. Graded degrees of severity of the brain malformation are also reflected in the highly variable craniofacial malformations associated with HPE. In addition, individuals with microforms of HPE, who usually have normal cognition and normal brain imaging, are at risk for having children with HPE. Some obligate carriers for HPE may not have any phenotypic abnormalities. Recurrent chromosomal rearrangements in individuals with HPE suggest loci containing genes important for brain development, and abnormalities in these genes may result in HPE. Recently, Sonic Hedgehog (SHH) was the first gene identified as causing HPE in humans. Proper function of SHH depends on cholesterol modification. Other candidate genes that may be involved in HPE include components of the SHH pathway, elements involved in cholesterol metabolism, and genes expressed in the developing forebrain.

Protein-Protein Interaction Network and Gene Ontology

NASA Astrophysics Data System (ADS)

Choi, Yunkyu; Kim, Seok; Yi, Gwan-Su; Park, Jinah

Evolution of computer technologies makes it possible to access a large amount and various kinds of biological data via internet such as DNA sequences, proteomics data and information discovered about them. It is expected that the combination of various data could help researchers find further knowledge about them. Roles of a visualization system are to invoke human abilities to integrate information and to recognize certain patterns in the data. Thus, when the various kinds of data are examined and analyzed manually, an effective visualization system is an essential part. One instance of these integrated visualizations can be combination of protein-protein interaction (PPI) data and Gene Ontology (GO) which could help enhance the analysis of PPI network. We introduce a simple but comprehensive visualization system that integrates GO and PPI data where GO and PPI graphs are visualized side-by-side and supports quick reference functions between them. Furthermore, the proposed system provides several interactive visualization methods for efficiently analyzing the PPI network and GO directedacyclic- graph such as context-based browsing and common ancestors finding.

Using neighborhood cohesiveness to infer interactions between protein domains.

PubMed

Segura, Joan; Sorzano, C O S; Cuenca-Alba, Jesus; Aloy, Patrick; Carazo, J M

2015-08-01

In recent years, large-scale studies have been undertaken to describe, at least partially, protein-protein interaction maps, or interactomes, for a number of relevant organisms, including human. However, current interactomes provide a somehow limited picture of the molecular details involving protein interactions, mostly because essential experimental information, especially structural data, is lacking. Indeed, the gap between structural and interactomics information is enlarging and thus, for most interactions, key experimental information is missing. We elaborate on the observation that many interactions between proteins involve a pair of their constituent domains and, thus, the knowledge of how protein domains interact adds very significant information to any interactomic analysis. In this work, we describe a novel use of the neighborhood cohesiveness property to infer interactions between protein domains given a protein interaction network. We have shown that some clustering coefficients can be extended to measure a degree of cohesiveness between two sets of nodes within a network. Specifically, we used the meet/min coefficient to measure the proportion of interacting nodes between two sets of nodes and the fraction of common neighbors. This approach extends previous works where homolog coefficients were first defined around network nodes and later around edges. The proposed approach substantially increases both the number of predicted domain-domain interactions as well as its accuracy as compared with current methods. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

The Protein Interaction Network of Bacteriophage Lambda with Its Host, Escherichia coli

PubMed Central

Blasche, Sonja; Wuchty, Stefan; Rajagopala, Seesandra V.

2013-01-01

Although most of the 73 open reading frames (ORFs) in bacteriophage λ have been investigated intensively, the function of many genes in host-phage interactions remains poorly understood. Using yeast two-hybrid screens of all lambda ORFs for interactions with its host Escherichia coli, we determined a raw data set of 631 host-phage interactions resulting in a set of 62 high-confidence interactions after multiple rounds of retesting. These links suggest novel regulatory interactions between the E. coli transcriptional network and lambda proteins. Targeted host proteins and genes required for lambda infection are enriched among highly connected proteins, suggesting that bacteriophages resemble interaction patterns of human viruses. Lambda tail proteins interact with both bacterial fimbrial proteins and E. coli proteins homologous to other phage proteins. Lambda appears to dramatically differ from other phages, such as T7, because of its unusually large number of modified and processed proteins, which reduces the number of host-virus interactions detectable by yeast two-hybrid screens. PMID:24049175

A mammalian germ cell-specific RNA-binding protein interacts with ubiquitously expressed proteins involved in splice site selection

NASA Astrophysics Data System (ADS)

Elliott, David J.; Bourgeois, Cyril F.; Klink, Albrecht; Stévenin, James; Cooke, Howard J.

2000-05-01

RNA-binding motif (RBM) genes are found on all mammalian Y chromosomes and are implicated in spermatogenesis. Within human germ cells, RBM protein shows a similar nuclear distribution to components of the pre-mRNA splicing machinery. To address the function of RBM, we have used protein-protein interaction assays to test for possible physical interactions between these proteins. We find that RBM protein directly interacts with members of the SR family of splicing factors and, in addition, strongly interacts with itself. We have mapped the protein domains responsible for mediating these interactions and expressed the mouse RBM interaction region as a bacterial fusion protein. This fusion protein can pull-down several functionally active SR protein species from cell extracts. Depletion and add-back experiments indicate that these SR proteins are the only splicing factors bound by RBM which are required for the splicing of a panel of pre-mRNAs. Our results suggest that RBM protein is an evolutionarily conserved mammalian splicing regulator which operates as a germ cell-specific cofactor for more ubiquitously expressed pre-mRNA splicing activators.

The regulation of tooth morphogenesis is associated with epithelial cell proliferation and the expression of Sonic hedgehog through epithelial-mesenchymal interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ishida, Kentaro; Murofushi, Mayumi; Nakao, Kazuhisa

2011-02-18

Research highlights: {yields} Bioengineered teeth regulated the contact area of epithelium and mesenchyme. {yields} The crown width is regulated by the contact area of the epithelium and mesenchyme. {yields} This regulation is associated with cell proliferation and Sonic hedgehog expression. {yields} The cusp number is correlated with the crown width of the bioengineered tooth. {yields} Cell proliferation and Shh expression areas regulate the tooth morphogenesis. -- Abstract: Ectodermal organs, such as the tooth, salivary gland, hair, and mammary gland, develop through reciprocal epithelial-mesenchymal interactions. Tooth morphologies are defined by the crown width and tooth length (macro-morphologies), and by the numbermore » and locations of the cusp and roots (micro-morphologies). In our current study, we report that the crown width of a bioengineered molar tooth, which was reconstructed using dissociated epithelial and mesenchymal cells via an organ germ method, can be regulated by the contact area between epithelial and mesenchymal cell layers. We further show that this is associated with cell proliferation and Sonic hedgehog (Shh) expression in the inner enamel epithelium after the germ stage has formed a secondary enamel knot. We also demonstrate that the cusp number is significantly correlated with the crown width of the bioengineered tooth. These findings suggest that the tooth micro-morphology, i.e. the cusp formation, is regulated after the tooth width, or macro-morphology, is determined. These findings also suggest that the spatiotemporal patterning of cell proliferation and the Shh expression areas in the epithelium regulate the crown width and cusp formation of the developing tooth.« less

Prediction of physical protein protein interactions

NASA Astrophysics Data System (ADS)

Szilágyi, András; Grimm, Vera; Arakaki, Adrián K.; Skolnick, Jeffrey

2005-06-01

Many essential cellular processes such as signal transduction, transport, cellular motion and most regulatory mechanisms are mediated by protein-protein interactions. In recent years, new experimental techniques have been developed to discover the protein-protein interaction networks of several organisms. However, the accuracy and coverage of these techniques have proven to be limited, and computational approaches remain essential both to assist in the design and validation of experimental studies and for the prediction of interaction partners and detailed structures of protein complexes. Here, we provide a critical overview of existing structure-independent and structure-based computational methods. Although these techniques have significantly advanced in the past few years, we find that most of them are still in their infancy. We also provide an overview of experimental techniques for the detection of protein-protein interactions. Although the developments are promising, false positive and false negative results are common, and reliable detection is possible only by taking a consensus of different experimental approaches. The shortcomings of experimental techniques affect both the further development and the fair evaluation of computational prediction methods. For an adequate comparative evaluation of prediction and high-throughput experimental methods, an appropriately large benchmark set of biophysically characterized protein complexes would be needed, but is sorely lacking.

Function of Brg1 Chromatin Remodeling Factor in Sonic Hedgehog-Dependent Medulloblastoma Initiation and Maintenance

DTIC Science & Technology

2014-10-01

Remodeling Factor in Sonic Hedgehog -Dependent Medulloblastoma Initiation and Maintenance PRINCIPAL INVESTIGATOR: Xuanming Shi CONTRACTING...Function of Brg1 Chromatin Remodeling Factor in Sonic Hedgehog -Dependent 5b. GRANT NUMBER W81XWH-12-1-0527 Medulloblastoma Initiation and Maintenance...medulloblastoma. 15. SUBJECT TERMS Medulloblastoma, Sonic Hedgehog , Chromatin remodeling, BAF complex, Brg1, mouse model of shh-subtype medulloblastoma

Changes in expression and secretion patterns of fibroblast growth factor 8 and Sonic Hedgehog signaling pathway molecules during murine neural stem/progenitor cell differentiation in vitro☆

PubMed Central

Lu, Jiang; Lu, Kehuan; Li, Dongsheng

2012-01-01

In the present study, we investigated the dynamic expression of fibroblast growth factor 8 and Sonic Hedgehog signaling pathway related factors in the process of in vitro hippocampal neural stem/progenitor cell differentiation from embryonic Sprague-Dawley rats or embryonic Kunming species mice, using fluorescent quantitative reverse transcription-PCR and western blot analyses. Results demonstrated that the dynamic expression of fibroblast growth factor 8 was similar to fibroblast growth factor receptor 1 expression but not to other fibroblast growth factor receptors. Enzyme-linked immunosorbent assay demonstrated that fibroblast growth factor 8 and Sonic Hedgehog signaling pathway protein factors were secreted by neural cells into the intercellular niche. Our experimental findings indicate that fibroblast growth factor 8 and Sonic Hedgehog expression may be related to the differentiation of neural stem/progenitor cells. PMID:25624789

Pyramidal neurons in the septal and temporal CA1 field of the human and hedgehog tenrec hippocampus.

PubMed

Liagkouras, Ioannis; Michaloudi, Helen; Batzios, Christos; Psaroulis, Dimitrios; Georgiadis, Marios; Künzle, Heinz; Papadopoulos, Georgios C

2008-07-07

The present study examines comparatively the cellular density of disector-counted/Nissl-stained CA1 pyramidal neurons and the morphometric characteristics (dendritic number/length, spine number/density and Sholl-counted dendritic branch points/20 microm) of the basal and apical dendritic systems of Golgi-impregnated CA1 neurons, in the septal and temporal hippocampus of the human and hedgehog tenrec brain. The obtained results indicate that in both hippocampal parts the cellular density of the CA1 pyramidal neurons is lower in human than in tenrec. However, while the human pyramidal cell density is higher in the septal hippocampal part than in the temporal one, in the tenrec the density of these cells is higher in the temporal part. The dendritic tree of the CA1 pyramidal cells, more developed in the septal than in temporal hippocampus in both species studied, is in general more complex in the human hippocampus. The basal and the apical dendritic systems exhibit species related morphometric differences, while dendrites of different orders exhibit differences in their number and length, and in their spine density. Finally, in both species, as well as hippocampal parts and dendritic systems, changes of dendritic morphometric features along ascending dendritic orders fluctuate in a similar way, as do the number of dendritic branch points in relation to the distance from the neuron soma.

Rigid-Docking Approaches to Explore Protein-Protein Interaction Space.

PubMed

Matsuzaki, Yuri; Uchikoga, Nobuyuki; Ohue, Masahito; Akiyama, Yutaka

Protein-protein interactions play core roles in living cells, especially in the regulatory systems. As information on proteins has rapidly accumulated on publicly available databases, much effort has been made to obtain a better picture of protein-protein interaction networks using protein tertiary structure data. Predicting relevant interacting partners from their tertiary structure is a challenging task and computer science methods have the potential to assist with this. Protein-protein rigid docking has been utilized by several projects, docking-based approaches having the advantages that they can suggest binding poses of predicted binding partners which would help in understanding the interaction mechanisms and that comparing docking results of both non-binders and binders can lead to understanding the specificity of protein-protein interactions from structural viewpoints. In this review we focus on explaining current computational prediction methods to predict pairwise direct protein-protein interactions that form protein complexes.

Eosinophilic leukemia in three African pygmy hedgehogs ( Atelerix albiventris) and validation of Luna stain.

PubMed

Martínez-Jiménez, David; Garner, Bridget; Coutermarsh-Ott, Sheryl; Burrell, Caitlin; Clark, Sabrina; Nabity, Mary; Díaz-Delgado, Josué; Rodrigues-Hoffmann, Aline; Zaks, Karen; Proença, Laila; Divers, Stephen; Saba, Corey; Cazzini, Paola

2017-03-01

Neoplasia is usually encountered in the African pygmy hedgehog at a mean age of 3.5 y, and malignancy is common. Myelogenous leukemias are rarely reported in hedgehogs. We describe 3 cases of eosinophilic leukemia in adult, middle-aged (mean age: 2.3 y) hedgehogs, for which prognosis appears grave. In 1 case, attempted treatment was unsuccessful, and in all 3 cases, the disease course was rapid and all died soon after diagnosis. Blood smear evaluation, along with complete blood count, was critical in making the diagnosis in all cases. Luna stain was validated and used to better visualize eosinophils in cytologic and histologic sections. Electron microscopy confirmed the presence of specific granules in hedgehog eosinophils.

Protein interactions and complexes in human microRNA biogenesis and function

PubMed Central

Perron, Marjorie P.; Provost, Patrick

2010-01-01

Encoded in the genome of most eukaryotes, microRNAs (miRNAs) have been proposed to regulate specifically up to 90% of human genes through a process known as miRNA-guided RNA silencing. The aim of this review is to present this process as the integration of a succession of specialized molecular machines exerting well defined functions. The nuclear microprocessor complex initially recognizes and processes its primary miRNA substrate into a miRNA precursor (pre-miRNA). This structure is then exported to the cytoplasm by the Exportin-5 complex where it is presented to the pre-miRNA processing complex. Following pre-miRNA conversion into a miRNA:miRNA* duplex, this complex is assembled into a miRNA-containing ribonucleoprotein (miRNP) complex, after which the miRNA strand is selected. The degree of complementarity of the miRNA for its messenger RNA (mRNA) target guides the recruitment of the miRNP complex. Initially repressing its translation, the miRNP-silenced mRNA is directed to the P-bodies, where the mRNA is either released from its inhibition upon a cellular signal and/or actively degraded. The potency and specificity of miRNA biogenesis and function rely on the distinct protein·protein, protein·RNA and RNA:RNA interactions found in different complexes, each of which fulfill a specific function in a well orchestrated process. PMID:17981733

A different kind of hedgehog pathway: tinea manus due to Trichophyton erinacei transmitted by an African pygmy hedgehog (Atelerix albiventris).

PubMed

Weishaupt, Julia; Kolb-Mäurer, Annette; Lempert, Sigrid; Nenoff, Pietro; Uhrlaß, Silke; Hamm, Henning; Goebeler, Matthias

2014-02-01

The unusual case of a 29-year-old woman with tinea manus caused by infection due to Trichophyton erinacei is described. The patient presented with marked erosive inflammation of the entire fifth finger of her right hand. Mycological and genomic diagnostics resulted in identification of T. erinacei as the responsible pathogen, which had been transmitted by a domestic African pygmy hedgehog, Atelerix albiventris. Upon prolonged treatment with topical and systemic antifungal agents skin lesions slowly resolved. This case illustrates that the increasingly popular keeping of extraordinary pets such as hedgehogs may bear the risk of infections with uncommon dermatophytes. © 2013 Blackwell Verlag GmbH.

Human Immunodeficiency Virus Type 1 Employs the Cellular Dynein Light Chain 1 Protein for Reverse Transcription through Interaction with Its Integrase Protein

PubMed Central

Jayappa, Kallesh Danappa; Ao, Zhujun; Wang, Xiaoxia; Mouland, Andrew J.; Shekhar, Sudhanshu; Yang, Xi

2015-01-01

ABSTRACT In this study, we examined the requirement for host dynein adapter proteins such as dynein light chain 1 (DYNLL1), dynein light chain Tctex-type 1 (DYNLT1), and p150Glued in early steps of human immunodeficiency virus type 1 (HIV-1) replication. We found that the knockdown (KD) of DYNLL1, but not DYNLT1 or p150Glued, resulted in significantly lower levels of HIV-1 reverse transcription in cells. Following an attempt to determine how DYNLL1 could impact HIV-1 reverse transcription, we detected the DYNLL1 interaction with HIV-1 integrase (IN) but not with capsid (CA), matrix (MA), or reverse transcriptase (RT) protein. Furthermore, by mutational analysis of putative DYNLL1 interaction motifs in IN, we identified the motifs 52GQVD and 250VIQD in IN as essential for DYNLL1 interaction. The DYNLL1 interaction-defective IN mutant HIV-1 (HIV-1INQ53A/Q252A) exhibited impaired reverse transcription. Through further investigations, we have also detected relatively smaller amounts of particulate CA in DYNLL1-KD cells or in infections with HIV-1INQ53A/Q252A mutant virus. Overall, our study demonstrates the novel interaction between HIV-1 IN and cellular DYNLL1 proteins and suggests the requirement of this virus-cell interaction for proper uncoating and efficient reverse transcription of HIV-1. IMPORTANCE Host cellular DYNLL1, DYNLT1, and p150Glued proteins have been implicated in the replication of several viruses. However, their roles in HIV-1 replication have not been investigated. For the first time, we demonstrated that during viral infection, HIV-1 IN interacts with DYNLL1, and their interaction was found to have a role in proper uncoating and efficient reverse transcription of HIV-1. Thus, interaction of IN and DYNLL1 may be a potential target for future anti-HIV therapy. Moreover, while our study has evaluated the involvement of IN in HIV-1 uncoating and reverse transcription, it also predicts a possible mechanism by which IN contributes to these early viral

The Hedgehog Signal Induced Modulation of Bone Morphogenetic Protein Signaling: An Essential Signaling Relay for Urinary Tract Morphogenesis

PubMed Central

Nakagata, Naomi; Miyagawa, Shinichi; Suzuki, Kentaro; Kitazawa, Sohei; Yamada, Gen

2012-01-01

Background Congenital diseases of the urinary tract are frequently observed in infants. Such diseases present a number of developmental anomalies such as hydroureter and hydronephrosis. Although some genetically-modified mouse models of growth factor signaling genes reproduce urinary phenotypes, the pathogenic mechanisms remain obscure. Previous studies suggest that a portion of the cells in the external genitalia and bladder are derived from peri-cloacal mesenchymal cells that receive Hedgehog (Hh) signaling in the early developmental stages. We hypothesized that defects in such progenitor cells, which give rise to urinary tract tissues, may be a cause of such diseases. Methodology/Principal Findings To elucidate the pathogenic mechanisms of upper urinary tract malformations, we analyzed a series of Sonic hedgehog (Shh) deficient mice. Shh−/− displayed hydroureter and hydronephrosis phenotypes and reduced expression of several developmental markers. In addition, we suggested that Shh modulation at an early embryonic stage is responsible for such phenotypes by analyzing the Shh conditional mutants. Tissue contribution assays of Hh-responsive cells revealed that peri-cloacal mesenchymal cells, which received Hh signal secreted from cloacal epithelium, could contribute to the ureteral mesenchyme. Gain- and loss-of-functional mutants for Hh signaling revealed a correlation between Hh signaling and Bone morphogenetic protein (Bmp) signaling. Finally, a conditional ablation of Bmp receptor type IA (BmprIA) gene was examined in Hh-responsive cell lineages. This system thus made it possible to analyze the primary functions of the growth factor signaling relay. The defective Hh-to-Bmp signaling relay resulted in severe urinary tract phenotypes with a decrease in the number of Hh-responsive cells. Conclusions/Significance This study identified the essential embryonic stages for the pathogenesis of urinary tract phenotypes. These results suggested that Hh

Study of human salivary proline-rich proteins interaction with food tannins.

PubMed

Soares, Susana; García-Estévez, Ignacio; Ferrer-Galego, Raúl; Brás, Natércia F; Brandão, Elsa; Silva, Mafalda; Teixeira, Natércia; Fonseca, Fátima; Sousa, Sérgio F; Ferreira-da-Silva, Frederico; Mateus, Nuno; de Freitas, Victor

2018-03-15

In this work, saturation transfer difference-NMR, isothermal microcalorimetry and molecular dynamics simulations have been used to study the individual interactions between basic, glycosylated and acidic proline-rich proteins (bPRPS, gPRPs, aPRPs) and P-B peptide with some representative food tannins [procyanidin B2, procyanidin B2 3'-O-gallate (B2g) and procyanidin trimer (catechin-4-8-catechin-4-8-catechin)]. Results showed that P-B peptide was in general the salivary protein (SP) with higher affinity whereas aPRPs showed lower affinity to the studied procyanidins. Moreover, B2g was the procyanidin with higher affinity for all SP. Hydrophobic and hydrogen bonds were present in all interactions but the major driving force depended on the procyanidin-SP pair. Furthermore, proline clusters or residues in their vicinity were identified as the probable sites of proteins for interaction with procyanidins. For bPRP and aPRP a significant change to less extended conformations was observed, while P-B peptide did not display any structural rearrangement upon procyanidins binding. Copyright © 2017. Published by Elsevier Ltd.

Direct bone morphogenetic protein 2 and Indian hedgehog gene transfer for articular cartilage repair using bone marrow coagulates.

PubMed

Sieker, J T; Kunz, M; Weißenberger, M; Gilbert, F; Frey, S; Rudert, M; Steinert, A F

2015-03-01

Bone morphogenetic protein 2 (BMP-2, encoded by BMP2) and Indian hedgehog protein (IHH, encoded by IHH) are well known regulators of chondrogenesis and chondrogenic hypertrophy. Despite being a potent chondrogenic factor BMP-2 was observed to induce chondrocyte hypertrophy in osteoarthritis (OA), growth plate cartilage and adult mesenchymal stem cells (MSCs). IHH might induce chondrogenic differentiation through different intracellular signalling pathways without inducing subsequent chondrocyte hypertrophy. The primary objective of this study is to test the efficacy of direct BMP2 and IHH gene delivery via bone marrow coagulates to influence histological repair cartilage quality in vivo. Vector-laden autologous bone marrow coagulates with 10(11) adenoviral vector particles encoding BMP2, IHH or the Green fluorescent protein (GFP) were delivered to 3.2 mm osteochondral defects in the trochlea of rabbit knees. After 13 weeks the histological repair cartilage quality was assessed using the ICRS II scoring system and the type II collagen positive area. IHH treatment resulted in superior histological repair cartilage quality than GFP controls in all of the assessed parameters (with P < 0.05 in five of 14 assessed parameters). Results of BMP2 treatment varied substantially, including severe intralesional bone formation in two of six joints after 13 weeks. IHH gene transfer is effective to improve repair cartilage quality in vivo, whereas BMP2 treatment, carried the risk intralesional bone formation. Therefore IHH protein can be considered as an attractive alternative candidate growth factor for further preclinical research and development towards improved treatments for articular cartilage defects. Copyright © 2014 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Exploiting Amino Acid Composition for Predicting Protein-Protein Interactions

PubMed Central

Roy, Sushmita; Martinez, Diego; Platero, Harriett; Lane, Terran; Werner-Washburne, Margaret

2009-01-01

Background Computational prediction of protein interactions typically use protein domains as classifier features because they capture conserved information of interaction surfaces. However, approaches relying on domains as features cannot be applied to proteins without any domain information. In this paper, we explore the contribution of pure amino acid composition (AAC) for protein interaction prediction. This simple feature, which is based on normalized counts of single or pairs of amino acids, is applicable to proteins from any sequenced organism and can be used to compensate for the lack of domain information. Results AAC performed at par with protein interaction prediction based on domains on three yeast protein interaction datasets. Similar behavior was obtained using different classifiers, indicating that our results are a function of features and not of classifiers. In addition to yeast datasets, AAC performed comparably on worm and fly datasets. Prediction of interactions for the entire yeast proteome identified a large number of novel interactions, the majority of which co-localized or participated in the same processes. Our high confidence interaction network included both well-studied and uncharacterized proteins. Proteins with known function were involved in actin assembly and cell budding. Uncharacterized proteins interacted with proteins involved in reproduction and cell budding, thus providing putative biological roles for the uncharacterized proteins. Conclusion AAC is a simple, yet powerful feature for predicting protein interactions, and can be used alone or in conjunction with protein domains to predict new and validate existing interactions. More importantly, AAC alone performs at par with existing, but more complex, features indicating the presence of sequence-level information that is predictive of interaction, but which is not necessarily restricted to domains. PMID:19936254