scholarly journals The binding of plakoglobin to desmosomal cadherins: patterns of binding sites and topogenic potential.

1996 ◽  
Vol 133 (2) ◽  
pp. 359-369 ◽  
Author(s):  
N A Chitaev ◽  
R E Leube ◽  
R B Troyanovsky ◽  
L G Eshkind ◽  
W W Franke ◽  
...  
Keyword(s):  
Recombinant Dna ◽  
Chimeric Protein ◽  
Binding Assays ◽  
Recombinant Dna Technology ◽  
Desmosomal Cadherins ◽  
Binding Domains ◽  
Transmembrane Glycoprotein ◽  
E Cadherin

Plakoglobin is the only protein that occurs in the cytoplasmic plaques of all known adhering junctions and has been shown to be crucially involved in the formation and maintenance of desmosomes anchoring intermediate-sized filaments (IFs) by its interaction with the desmosomal cadherins, desmoglein (Dsg), and desmocollin (Dsc). This topogenic importance of plakoglobin is now directly shown in living cells as well as in binding assays in vitro. We show that, in transfected human A-431 carcinoma cells, a chimeric protein combining the vesicle-forming transmembrane glycoprotein synaptophysin, with the complete human plakoglobin sequence, is sorted to small vesicles many of which associate with desmosomal plaques and their attached IFs. Immunoprecipitation experiments have further revealed that the chimeric plakoglobin-containing transmembrane molecules of these vesicles are tightly bound to Dsg and Dsc but not to endogenous plakoglobin, thus demonstrating that the binding of plakoglobin to desmosomal cadherins does not require its soluble state and is strong enough to attach large structures such as vesicles to desmosomes. To identify the binding domains and the mechanisms involved in the interaction of plakoglobin with desmosomal cadherins, we have developed direct binding assays in vitro in which plakoglobin or parts thereof, produced by recombinant DNA technology in E. coli, are exposed to molecules containing the "C-domains" of several cadherins. These assays have shown that plakoglobin associates most tightly with the C-domain of Dsg, to a lesser degree with that of Dsc and only weakly with the C-domain of E-cadherin. Three separate segments of plakoglobin containing various numbers of the so-called arm repeats exhibit distinct binding to the desmosomal cadherins comparable in strength to that of the entire molecule. The binding pattern of plakoglobin segments in vitro is compared with that in vivo. Paradoxically, in vitro some internal plakoglobin fragments bind even better to the C-domain of E-cadherin than the entire molecule, indicating that elements exist in native plakoglobin that interfere with the interaction of this protein with its various cadherin partners.

scholarly journals Inhibition of Fungal β-1,3-Glucan Synthase and Cell Growth by HM-1 Killer Toxin Single-Chain Anti-Idiotypic Antibodies

2006 ◽  
Vol 50 (9) ◽  
pp. 3090-3097 ◽  
Author(s):  
Dakshnamurthy Selvakumar ◽  
Masahiko Miyamoto ◽  
Yasuhiro Furuichi ◽  
Tadazumi Komiyama
Keyword(s):  
Recombinant Dna ◽  
Killer Toxin ◽  
Binding Assays ◽  
Single Chain ◽  
Recombinant Dna Technology ◽  
Surface Plasmon Resonance Analysis ◽  
Glucan Synthase ◽  
Recombinant Scfv ◽  
Neutralizing Monoclonal Antibody

ABSTRACT Single-chain variable-fragment (scFv) anti-idiotypic antibodies of an HM-1 killer toxin (HM-1) from the yeast Williopsis saturnus var. mrakii IFO 0895 have been produced by recombinant DNA technology from the splenic lymphocytes of mice immunized by idiotypic vaccination with a neutralizing monoclonal antibody (nMAb-KT). The fungicidal activity of scFv anti-idiotypic antibodies against the isolates of four Candida species was assessed by MIC analysis. scFv antibodies were fungicidal at concentrations of 1.56 to 12.5 μg/ml in vitro against four Candida species. The scFv antibodies exerted a strong candidacidal activity in vitro, with 50% inhibitory concentration (IC50) values ranging from 7.3 × 10−8 to 16.0 × 10−8 M, and were neutralized by adsorption with nMAb-KT. Furthermore, all scFv antibodies effectively inhibited fungal β-1,3-glucan synthase activity in vitro, with IC50 values ranging from 2.0 × 10−8 to 22.7 × 10−8 M, values which almost coincide with the values that are inhibitory to the growth of fungal cells. Binding assays showed that the scFv antibodies specifically bind to nMAb-KT, and this binding pattern was confirmed by surface plasmon resonance analysis. The binding ability was further demonstrated by the competition observed between scFv antibodies and HM-1 to bind nMAb-KT. To the best of our knowledge, this is the first study to show that an antifungal anti-idiotypic antibody, in the form of recombinant scFv, potentially inhibits β-1,3-glucan synthase activity.

Interferons in the treatment of human cancer.

1984 ◽  
Vol 2 (4) ◽  
pp. 336-352 ◽  
Author(s):  
J M Kirkwood ◽  
M S Ernstoff
Keyword(s):  
Recombinant Dna ◽  
Human Cancer ◽  
Clinical Results ◽  
Common Species ◽  
Current Phase ◽  
Recombinant Dna Technology ◽  
Disease States ◽  
Wide Range

The interferons are the best known of biologic antineoplastic agents. Progress with the clinical application of interferons to cancer has been slow and complicated by the need for attention to a new spectrum of therapeutic and toxic effects manifest by the interferons. This summary of current phase I and II trial results with the interferons establishes their clinical potential. The maximally tolerated dosages of the most common species of interferon alpha produced in eukaryotic cells as well as by recombinant DNA technology in bacteria are now described in a variety of different disease states. "Naturally" produced eukaryotic as well as bacterially synthesized interferons have a similar, wide range of biologic effects in vitro and in vivo. Antiviral, antiproliferative, immunologic, and enzymologic functions of the interferons relevant to antineoplastic functions are under study. Knowledge of these mechanisms should improve the clinical results obtained in human cancer. Species and subspecies differences in the activity of interferons may lead to selective use of the pure interferon subspecies, alone or in combination. The use of the interferons and other antineoplastic biologics, such as antibody or chemotherapy, are subsequent goals that are now on the horizon.

scholarly journals Analysis of the Dynein-Dynactin Interaction In Vitro and In Vivo

2003 ◽  
Vol 14 (12) ◽  
pp. 5089-5097 ◽  
Author(s):  
Stephen J. King ◽  
Christa L. Brown ◽  
Kerstin C. Maier ◽  
Nicholas J. Quintyne ◽  
Trina A. Schroer
Keyword(s):  
Cytoplasmic Dynein ◽  
Binding Assays ◽  
Microtubule Organization ◽  
Binding Domains ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Dynein Intermediate Chain ◽  
Transient Overexpression

Cytoplasmic dynein and dynactin are megadalton-sized multisubunit molecules that function together as a cytoskeletal motor. In the present study, we explore the mechanism of dynein-dynactin binding in vitro and then extend our findings to an in vivo context. Solution binding assays were used to define binding domains in the dynein intermediate chain (IC) and dynactin p150Glued subunit. Transient overexpression of a series of fragments of the dynein IC was used to determine the importance of this subunit for dynein function in mammalian tissue culture cells. Our results suggest that a functional dynein-dynactin interaction is required for proper microtubule organization and for the transport and localization of centrosomal components and endomembrane compartments. The dynein IC fragments have different effects on endomembrane localization, suggesting that different endomembranes may bind dynein via distinct mechanisms.

The Parkes lecture: controlled ovarian stimulation in women

Reproduction ◽  
2000 ◽  
pp. 201-210 ◽  
Author(s):  
SG Hillier
Keyword(s):  
Ovarian Stimulation ◽  
Ovarian Function ◽  
Recombinant Dna ◽  
New Drugs ◽  
Controlled Ovarian Stimulation ◽  
Recombinant Dna Technology ◽  
Vitro Fertilization ◽  
Ovarian Folliculogenesis

Recent advances in knowledge of the endocrine and paracrine mechanisms that regulate human ovarian folliculogenesis have been parallelled by the introduction into clinical practice of new drugs that can be used safely and effectively to stimulate ovarian function in infertile women. Most notably, recombinant DNA technology has been applied to the production of molecularly pure forms of the gonadotrophins, FSH and LH, opening the way to the development of improved strategies for manipulating the ovarian paracrine system. The clinical objectives of controlled ovarian stimulation fall into two categories, depending on patient needs: (1) induction of multiple follicles from which mature oocytes can be harvested for use in assisted reproduction protocols such as in vitro fertilization and embryo transfer; or (2) induction of spontaneous ovulation of a single mature follicle so that conception might occur in vivo. This review summarizes the physiological principles upon which the use of gonadotrophins for clinical purposes is based, highlighting new opportunities for improved treatment as a result of the availability of recombinant FSH and LH.

scholarly journals Immunoregulatory role of transforming growth factor beta (TGF-beta) in development of killer cells: comparison of active and latent TGF-beta 1.

1990 ◽  
Vol 172 (6) ◽  
pp. 1777-1784 ◽  
Author(s):  
S C Wallick ◽  
I S Figari ◽  
R E Morris ◽  
A D Levinson ◽  
M A Palladino
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Recombinant Dna ◽  
Chinese Hamster ◽  
Tgf Beta ◽  
Recombinant Dna Technology ◽  
Growth Factor Beta

Using recombinant DNA technology, we have generated Chinese hamster ovary (CHO) cell lines that synthesize latent transforming growth factor beta 1 (TGF-beta 1) to study immune regulation by TGF-beta 1. In vitro, latent TGF-beta 1 synthesized by transfectants or added exogenously as a purified complex after activation inhibited CTL generation to a similar extent as seen with acid-activated recombinant human (rHu) TGF-beta 1. In vivo, serum from nu/nu mice bearing CHO/TGF-beta 1 tumors contained significant levels of latent TGF-beta 1 in addition to depressed natural killer (NK) activity in spleens which paralleled that seen in C3H/HeJ mice treated with acid-activated rHuTGF-beta 1. rHuTGF-beta 1 treatment of mice receiving heart allografts resulted in significant enhancement of organ graft survival. Because of possible regulated tissue-specific activation, administration of latent rather than active TGF-beta may provide a better route to deliver this powerful immunosuppressive agent in vivo.

scholarly journals H-Ras Activation Promotes Cytoplasmic Accumulation and Phosphoinositide 3-Oh Kinase Association of β-Catenin in Epidermal Keratinocytes

1999 ◽  
Vol 146 (5) ◽  
pp. 967-980 ◽  
Author(s):  
Jesús Espada ◽  
Mirna Pérez-Moreno ◽  
Vania M.M. Braga ◽  
Pablo Rodriguez-Viciana ◽  
Amparo Cano
Keyword(s):  
Glycogen Synthase ◽  
Strong Association ◽  
Direct Interaction ◽  
Epidermal Keratinocytes ◽  
Binding Assays ◽  
Adenomatous Polyposis Coli Protein ◽  
Ras Activation ◽  
E Cadherin

The mechanisms underlying downregulation of the cadherin/catenin complexes and β-catenin signaling during tumor progression are not fully understood. We have analyzed the effect of oncogenic H-Ras on E-cadherin/catenin complex formation/stabilization and β-catenin distribution in epidermal keratinocytes. Microinjection or stable expression of V12Ras into keratinocytes promotes the loss of E-cadherin and α-catenin and relocalization of β-catenin to the cytoplasm and nucleus. Moreover, these effects are dependent on PI3K (phosphoinositide 3-OH kinase) activity. Interestingly, a strong association of p85α and p110α subunits of PI3K with β-catenin is induced in V12Ras-expressing keratinocytes, and in vitro binding assays show a direct interaction between β-catenin and p85α. Overexpression of either V12Ras or constitutively active p110α induces metabolic stabilization of β-catenin and promotes its accumulation in cytoplasmic and nuclear pools. In addition, the interaction of β-catenin with the adenomatous polyposis coli protein is blocked in V12Ras and p110α transformants though no changes in glycogen synthase kinase 3 β activity could be detected. Nevertheless, in V12Ras transformants the in vivo phosphorylation of β-catenin in Ser residues is strongly decreased. These results indicate that H-Ras activation induces the relocalization and cytoplasmic stabilization of β-catenin by a mechanism involving its interaction with PI3K.

scholarly journals An epilepsy-causing mutation leads to co-translational misfolding of the Kv7.2 channel

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Janire Urrutia ◽  
Alejandra Aguado ◽  
Carolina Gomis-Perez ◽  
Arantza Muguruza-Montero ◽  
Oscar R. Ballesteros ◽  
...  
Keyword(s):  
Structural Instability ◽  
Binding Assays ◽  
Iq Motif ◽  
Channel Function ◽  
Human Epilepsy ◽  
Pathogenic Variants ◽  
In Silico Studies ◽  
Nascent Chain

Abstract Background The amino acid sequence of proteins generally carries all the necessary information for acquisition of native conformations, but the vectorial nature of translation can additionally determine the folding outcome. Such consideration is particularly relevant in human diseases associated to inherited mutations leading to structural instability, aggregation, and degradation. Mutations in the KCNQ2 gene associated with human epilepsy have been suggested to cause misfolding of the encoded Kv7.2 channel. Although the effect on folding of mutations in some domains has been studied, little is known of the way pathogenic variants located in the calcium responsive domain (CRD) affect folding. Here, we explore how a Kv7.2 mutation (W344R) located in helix A of the CRD and associated with hereditary epilepsy interferes with channel function. Results We report that the epilepsy W344R mutation within the IQ motif of CRD decreases channel function, but contrary to other mutations at this site, it does not impair the interaction with Calmodulin (CaM) in vitro, as monitored by multiple in vitro binding assays. We find negligible impact of the mutation on the structure of the complex by molecular dynamic computations. In silico studies revealed two orientations of the side chain, which are differentially populated by WT and W344R variants. Binding to CaM is impaired when the mutated protein is produced in cellulo but not in vitro, suggesting that this mutation impedes proper folding during translation within the cell by forcing the nascent chain to follow a folding route that leads to a non-native configuration, and thereby generating non-functional ion channels that fail to traffic to proper neuronal compartments. Conclusions Our data suggest that the key pathogenic mechanism of Kv7.2 W344R mutation involves the failure to adopt a configuration that can be recognized by CaM in vivo but not in vitro.

scholarly journals HDAC4 promotes nasopharyngeal carcinoma progression and serves as a therapeutic target

2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Chun Cheng ◽  
Jun Yang ◽  
Si-Wei Li ◽  
Guofu Huang ◽  
Chenxi Li ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Tumor Growth ◽  
Therapeutic Target ◽  
Histone Deacetylases ◽  
Migration And Invasion ◽  
Poor Overall Survival ◽  
Mesenchymal Transition ◽  
E Cadherin

AbstractHistone deacetylases (HDACs) are involved in tumor progression, and some have been successfully targeted for cancer therapy. The expression of histone deacetylase 4 (HDAC4), a class IIa HDAC, was upregulated in our previous microarray screen. However, the role of HDAC4 dysregulation and mechanisms underlying tumor growth and metastasis in nasopharyngeal carcinoma (NPC) remain elusive. Here, we first confirmed that the HDAC4 levels in primary and metastatic NPC tissues were significantly increased compared with those in normal nasopharyngeal epithelial tissues and found that high HDAC4 expression predicted a poor overall survival (OS) and progression-free survival (PFS). Functionally, HDAC4 accelerated cell cycle G1/S transition and induced the epithelial-to-mesenchymal transition to promote NPC cell proliferation, migration, and invasion in vitro, as well as tumor growth and lung metastasis in vivo. Intriguingly, knockdown of N-CoR abolished the effects of HDAC4 on the invasion and migration abilities of NPC cells. Mechanistically, HDAC3/4 binds to the E-cadherin promoter to repress E-cadherin transcription. We also showed that the HDAC4 inhibitor tasquinimod suppresses tumor growth in NPC. Thus, HDAC4 may be a potential diagnostic marker and therapeutic target in patients with NPC.

scholarly journals Genome-Wide Binding Analyses of HOXB1 Revealed a Novel DNA Binding Motif Associated with Gene Repression

2021 ◽  
Vol 9 (1) ◽  
pp. 6
Author(s):  
Narendra Pratap Singh ◽  
Bony De Kumar ◽  
Ariel Paulson ◽  
Mark E. Parrish ◽  
Carrie Scott ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Embryonic Stem ◽  
Binding Motif ◽  
Binding Assays ◽  
Histone Marks ◽  
Genome Wide ◽  
Hox Cofactors

Knowledge of the diverse DNA binding specificities of transcription factors is important for understanding their specific regulatory functions in animal development and evolution. We have examined the genome-wide binding properties of the mouse HOXB1 protein in embryonic stem cells differentiated into neural fates. Unexpectedly, only a small number of HOXB1 bound regions (7%) correlate with binding of the known HOX cofactors PBX and MEIS. In contrast, 22% of the HOXB1 binding peaks display co-occupancy with the transcriptional repressor REST. Analyses revealed that co-binding of HOXB1 with PBX correlates with active histone marks and high levels of expression, while co-occupancy with REST correlates with repressive histone marks and repression of the target genes. Analysis of HOXB1 bound regions uncovered enrichment of a novel 15 base pair HOXB1 binding motif HB1RE (HOXB1 response element). In vitro template binding assays showed that HOXB1, PBX1, and MEIS can bind to this motif. In vivo, this motif is sufficient for direct expression of a reporter gene and over-expression of HOXB1 selectively represses this activity. Our analyses suggest that HOXB1 has evolved an association with REST in gene regulation and the novel HB1RE motif contributes to HOXB1 function in part through a repressive role in gene expression.

Sign in / Sign up

Export Citation Format

Share Document