scholarly journals Engineering Disulphide-Free Autonomous Antibody VH Domains to modulate intracellular pathways

2021 ◽  
Author(s):  
Yuri Frosi ◽  
Shimin Jiang ◽  
Shimin Jiang ◽  
Siti Radhiah Ramlan ◽  
Kelly Hew Hew ◽  
...  
Keyword(s):  
Cellular Proliferation ◽  
Tumour Therapy ◽  
High Affinity ◽  
Eif4f Complex ◽  
Expression Studies ◽  
Interaction Interface ◽  
Intracellular Expression ◽  
Intracellular Pathways ◽  
Vh Domain ◽  
Related Proteins

Abstract An attractive approach to target intracellular macromolecular interfaces is to design small high affinity proteins. In this manuscript a stable, autonomous, human derived non-immunogenic, disulphide-free VH domain, has been engineered for intracellular expression studies. VH domains can be designed to possess a large dynamic repertoire of binders, as opposed to other scaffolds types that are highly rigid and possess fewer sites of random variation. Picomolar inhibitors were identified using phage display against the eIF4F complex, which is commonly hyper-activated in many cancers. These molecules were also shown to impair cellular proliferation and to reduce the expression of malignancy related proteins. Structural characterization elucidated that these VH domains bound eIF4E at the eIF4G interaction interface via a novel binding pose. Molecules able to mimic this pose and interfere with the eIF4F complex are potentially important for wide-ranging tumour therapy applications.

scholarly journals Constitutive and Regulated Shedding of Soluble FGF Receptors Releases Biologically Active Inhibitors of FGF-2

2021 ◽  
Vol 22 (5) ◽  
pp. 2712
Author(s):  
Anne Hanneken ◽  
Maluz Mercado ◽  
Pamela Maher
Keyword(s):  
Cell Surface ◽  
Ligand Binding ◽  
Cellular Proliferation ◽  
Biological Activities ◽  
Biological Properties ◽  
Matrix Metalloproteases ◽  
Biologically Active ◽  
Ectodomain Shedding ◽  
High Affinity

The identification of soluble fibroblast growth factor (FGF) receptors in blood and the extracellular matrix has led to the prediction that these proteins modulate the diverse biological activities of the FGF family of ligands in vivo. A recent structural characterization of the soluble FGF receptors revealed that they are primarily generated by proteolytic cleavage of the FGFR-1 ectodomain. Efforts to examine their biological properties are now focused on understanding the functional consequences of FGFR-1 ectodomain shedding and how the shedding event is regulated. We have purified an FGFR-1 ectodomain that is constitutively cleaved from the full-length FGFR-1(IIIc) receptor and released into conditioned media. This shed receptor binds FGF-2; inhibits FGF-2-induced cellular proliferation; and competes with high affinity, cell surface FGF receptors for ligand binding. FGFR-1 ectodomain shedding downregulates the number of high affinity receptors from the cell surface. The shedding mechanism is regulated by ligand binding and by activators of PKC, and the two signaling pathways appear to be independent of each other. Deletions and substitutions at the proposed cleavage site of FGFR-1 do not prevent ectodomain shedding. Broad spectrum inhibitors of matrix metalloproteases decrease FGFR-1 ectodomain shedding, suggesting that the enzyme responsible for constitutive, ligand-activated, and protein kinase C-activated shedding is a matrix metalloprotease. In summary, shedding of the FGFR-1 ectodomain is a highly regulated event, sharing many features with a common system that governs the release of diverse membrane proteins from the cell surface. Most importantly, the FGFR ectodomains are biologically active after shedding and are capable of functioning as inhibitors of FGF-2.

scholarly journals A Na+:H+ Antiporter and a Molybdate Transporter Are Essential for Arsenite Oxidation in Agrobacterium tumefaciens

2006 ◽  
Vol 188 (4) ◽  
pp. 1577-1584 ◽  
Author(s):  
Des R. Kashyap ◽  
Lina M. Botero ◽  
Corinne Lehr ◽  
Daniel J. Hassett ◽  
Timothy R. McDermott
Keyword(s):  
Gene Expression ◽  
Agrobacterium Tumefaciens ◽  
Genetic Determinants ◽  
High Affinity ◽  
Transposon Insertion ◽  
Expression Studies ◽  
Soil Isolate ◽  
Show Evidence ◽  
Gene Expression Studies ◽  
Membrane Gene

ABSTRACT Transposon Tn5-B22 mutagenesis was used to identify genetic determinants required for arsenite [As(III)] oxidation in an Agrobacterium tumefaciens soil isolate, strain 5A. In one mutant, the transposon interrupted modB, which codes for the permease component of a high-affinity molybdate transporter. In a second mutant, the transposon insertion occurred in mrpB, which is part of a seven-gene operon encoding an Mrp-type Na+:H+ antiporter complex. Complementation experiments with mod and mrp operons PCR cloned from the genome-sequenced A. tumefaciens strain C58 resulted in complementation back to an As(III)-oxidizing phenotype, confirming that these genes encode activities essential for As(III) oxidation in this strain of A. tumefaciens. As expected, the mrp mutant was extremely sensitive to NaCl and LiCl, indicating that the Mrp complex in A. tumefaciens is involved in Na+ circulation across the membrane. Gene expression studies (lacZ reporter and reverse transcriptase PCR experiments) failed to show evidence of transcriptional regulation of the mrp operon in response to As(III) exposure, whereas expression of the mod operon was found to be up-regulated by As(III) exposure. In each mutant, the loss of As(III)-oxidizing capacity resulted in conversion to an arsenate [As(V)]-reducing phenotype. Neither mutant was more sensitive to As(III) than the parental strain.

scholarly journals Hepatic transcription of the acute-phase alpha 1-inhibitor III gene is controlled by a novel combination of cis-acting regulatory elements.

1990 ◽  
Vol 10 (7) ◽  
pp. 3483-3491 ◽  
Author(s):  
L J Abraham ◽  
A D Bradshaw ◽  
B R Shiels ◽  
W Northemann ◽  
G Hudson ◽  
...  
Keyword(s):  
Binding Sites ◽  
Transcriptional Start Site ◽  
Regulatory Elements ◽  
Nuclear Factor ◽  
Base Pairs ◽  
Cis Acting ◽  
Expression Studies ◽  
Positive Regulators ◽  
Flanking Region ◽  
Related Proteins

mRNA coding for the abundant broad-range plasma proteinase inhibitor alpha 1-inhibitor III (alpha 1I3) was detected only in rat liver, while mRNA for the related proteins alpha 1-macroglobulin and alpha 2-macroglobulin was also found in a variety of nonhepatic tissues. cis-Acting control elements necessary for the hepatic transcription of alpha 1I3 were mapped by transfection and expression studies of control-region constructs in cultured hepatic and nonhepatic cells. The promoter-proximal 5'-flanking region contained four control elements, I to IV, located between -109 and -196 base pairs upstream of the transcriptional start site relevant for the hepatic transcription of this gene. Elements II and III were essential, and I and IV exerted strong modulatory effects. Elements I to III acted as positive regulators, and IV acted as a negative element. Element II contained the sequence TGGCA and is probably a binding site for a nuclear factor related to the known transcription factor NF1. The other three elements did not resemble consensus binding sites for known transcription factors that are involved in the hepatocyte-specific transcription of other well-characterized plasma protein genes, such as the prototype factor HNF-1. Thus, the alpha 1I3 gene achieves its highly hepatocyte-specific transcription through a novel combination of cis-acting control elements and trans-acting factors.

scholarly journals Foetal-calf serum stimulates a pertussis-toxin-sensitive high-affinity GTPase activity in rat glioma C6 BU1 cells

1987 ◽  
Vol 245 (2) ◽  
pp. 501-505 ◽  
Author(s):  
G Milligan
Keyword(s):  
Pertussis Toxin ◽  
Cellular Proliferation ◽  
Calf Serum ◽  
Foetal Calf Serum ◽  
Gtpase Activity ◽  
Guanine Nucleotide Binding Protein ◽  
Rat Glioma ◽  
High Affinity ◽  
Glioma C6 ◽  
Guanine Nucleotide Binding

Cellular proliferation of rat glioma C6 BU1 cells in tissue culture is dependent on the presence of either calf or foetal-calf serum in the medium. Foetal-calf serum stimulated a high-affinity GTPase in membranes derived from C6 BU1 cells. Pretreatment of the cells with pertussis toxin decreased the high-affinity GTPase activity substantially, and attenuated the foetal-calf-serum-stimulated increase in this GTPase activity. Cholera toxin, in contrast, did not modulate the response to foetal-calf serum. Foetal-calf serum did not inhibit adenylate cyclase activity in membranes of these cells, indicating that the G-protein that was stimulated by foetal-calf serum was not Gi (the inhibitory one). Although the nature of the specific component of foetal-calf serum responsible for this pertussis-toxin-sensitive receptor-mediated stimulation of high-affinity GTPase activity has not been identified, it was mimicked neither by bombesin, which can stimulate inositol phospholipid turnover via a guanine nucleotide binding protein, nor by platelet-derived growth factor, which is present in substantial concentrations in foetal-calf serum. This report represents the first demonstration of a pertussis-toxin-substrate-mediated response in this cell line and provides further evidence that G-proteins other than Gi can be functionally inactivated by pertussis toxin.

Expression studies of Nrt2:1Np, a putative high-affinity nitrate transporter: evidence for its role in nitrate uptake

1998 ◽  
Vol 14 (6) ◽  
pp. 723-731 ◽  
Author(s):  
Anne Krapp ◽  
Vincent Fraisier ◽  
Wolf-Rudiger Scheible ◽  
Alberto Quesada ◽  
Alain Gojon ◽  
...  
Keyword(s):  
Nitrate Uptake ◽  
Nitrate Transporter ◽  
High Affinity ◽  
Expression Studies

Hepatic transcription of the acute-phase alpha 1-inhibitor III gene is controlled by a novel combination of cis-acting regulatory elements

1990 ◽  
Vol 10 (7) ◽  
pp. 3483-3491
Author(s):  
L J Abraham ◽  
A D Bradshaw ◽  
B R Shiels ◽  
W Northemann ◽  
G Hudson ◽  
...  
Keyword(s):  
Binding Sites ◽  
Transcriptional Start Site ◽  
Regulatory Elements ◽  
Nuclear Factor ◽  
Base Pairs ◽  
Cis Acting ◽  
Expression Studies ◽  
Positive Regulators ◽  
Flanking Region ◽  
Related Proteins

mRNA coding for the abundant broad-range plasma proteinase inhibitor alpha 1-inhibitor III (alpha 1I3) was detected only in rat liver, while mRNA for the related proteins alpha 1-macroglobulin and alpha 2-macroglobulin was also found in a variety of nonhepatic tissues. cis-Acting control elements necessary for the hepatic transcription of alpha 1I3 were mapped by transfection and expression studies of control-region constructs in cultured hepatic and nonhepatic cells. The promoter-proximal 5'-flanking region contained four control elements, I to IV, located between -109 and -196 base pairs upstream of the transcriptional start site relevant for the hepatic transcription of this gene. Elements II and III were essential, and I and IV exerted strong modulatory effects. Elements I to III acted as positive regulators, and IV acted as a negative element. Element II contained the sequence TGGCA and is probably a binding site for a nuclear factor related to the known transcription factor NF1. The other three elements did not resemble consensus binding sites for known transcription factors that are involved in the hepatocyte-specific transcription of other well-characterized plasma protein genes, such as the prototype factor HNF-1. Thus, the alpha 1I3 gene achieves its highly hepatocyte-specific transcription through a novel combination of cis-acting control elements and trans-acting factors.

scholarly journals The Aminoterminal Insulin-Like Growth Factor (IGF) Binding Domain of IGF Binding Protein-3 Cannot Be Functionally Substituted by the Structurally Homologous Domain of CCN3

Endocrinology ◽  
2006 ◽  
Vol 147 (11) ◽  
pp. 5268-5274 ◽  
Author(s):  
Xiaolang Yan ◽  
Robert C. Baxter ◽  
Bernard Perbal ◽  
Sue M. Firth
Keyword(s):  
Growth Factor ◽  
Binding Site ◽  
Tissue Growth ◽  
Ccn Proteins ◽  
Homologous Proteins ◽  
Direct Role ◽  
High Affinity ◽  
Igf Binding ◽  
Related Proteins ◽  
Igfbp 3

IGF binding proteins (IGFBPs) are a family of structurally homologous proteins that bind IGFs with high affinities and can modulate IGF activity. The IGF binding site has been shown to comprise residues in both the aminoterminal and carboxyterminal domains. In recent years several proteins including members of the CCN (connective tissue growth factor, Cyr61, and nephroblastoma overexpressed) family were recognized as having structural homology in their aminoterminal domains to the IGFBPs. Despite their low or undetectable IGF binding ability, a proposal was made to rename them as IGFBP-related proteins. To test whether the aminoterminal domain of a CCN protein can fulfill the high-affinity IGF binding function of an IGFBP, we created a chimera in which the aminoterminal domain of IGFBP-3 was substituted with the aminoterminal domain of CCN3 (previously known as Nov). The CCN3-IGFBP-3 chimera bound IGFs and inhibited IGF activity very weakly, similar to CCN3 itself. Although structurally similar, the aminoterminal domain of CCN3 is unable to replace the aminoterminal domain of IGFBP-3 in forming a high-affinity IGF-binding site. These results argue against a direct role of CCN3 in the regulation of IGF bioavailability and indicate that the nomenclature of IGFBP-related proteins (which implies functional relationship to the classical IGFBPs) is inappropriate for CCN proteins.

scholarly journals Calculating metalation in cells reveals CobW acquires CoII for vitamin B12 biosynthesis while related proteins prefer ZnII

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Tessa R. Young ◽  
Maria Alessandra Martini ◽  
Andrew W. Foster ◽  
Arthur Glasfeld ◽  
Deenah Osman ◽  
...  
Keyword(s):  
Free Energy ◽  
Vitamin B12 ◽  
Vitamin B ◽  
Free Energies ◽  
High Affinity ◽  
High Affinity Site ◽  
Metal Sensors ◽  
Related Proteins ◽  
Intracellular Milieu

AbstractProtein metal-occupancy (metalation) in vivo has been elusive. To address this challenge, the available free energies of metals have recently been determined from the responses of metal sensors. Here, we use these free energy values to develop a metalation-calculator which accounts for inter-metal competition and changing metal-availabilities inside cells. We use the calculator to understand the function and mechanism of GTPase CobW, a predicted CoII-chaperone for vitamin B12. Upon binding nucleotide (GTP) and MgII, CobW assembles a high-affinity site that can obtain CoII or ZnII from the intracellular milieu. In idealised cells with sensors at the mid-points of their responses, competition within the cytosol enables CoII to outcompete ZnII for binding CobW. Thus, CoII is the cognate metal. However, after growth in different [CoII], CoII-occupancy ranges from 10 to 97% which matches CobW-dependent B12 synthesis. The calculator also reveals that related GTPases with comparable ZnII affinities to CobW, preferentially acquire ZnII due to their relatively weaker CoII affinities. The calculator is made available here for use with other proteins.

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