scholarly journals The Synthesis of Protein Polymer Conjugates using the Human Regulatory Protein Galectin-3

2020 ◽  
Vol 21 (2) ◽  
Author(s):  
Ling Lin ◽  
Amanda M. Pritzlaff ◽  
Haillie-Ann C. Lower ◽  
Daniel A. Savin
Keyword(s):  
Ligand Binding ◽  
Regulatory Protein ◽  
Site Directed Mutagenesis ◽  
Galectin 3 ◽  
Self Association ◽  
Lectin Protein ◽  
And Control ◽  
Cancerous Cells

Galectin-3 (gal3) is a human lectin protein that is known to interact with extracellular matrix proteins by regulating functions in both healthy and cancerous cells. The goal of this project is to conjugate polymers to gal3 to better study and control its functions in vitro. We hypothesize that a covalently attached polymer will sterically modulate gal3 function. In the project, we created two protein variants with polymer-reactive handles. The first construct is similar to wild-type gal3 with a cysteine in place of the 6th serine (S6C) which was created by site-directed mutagenesis (SDM). Maleimide-terminated polyethylene oxide (PEO, 5000 g/mol) was then attached to this mutant via thiol-Michael addition at the cysteine site. Attachment of polymer to the unstructured N-terminal domain (NTD) may increase the binding of the protein by sterically pulling the NTD away from the carbohydrate recognition domain (CRD). In addition, the NTD, which is implicated in undesired self-association, was removed for the second construct. The gal3 CRD only construct is shown to have a higher solubility in solution and an increased ligand-binding affinity. Ultimately, the two unique constructs will help us understand the structural role of the NTD in gal3 ligand-binding and self-association.

scholarly journals The role of Val68(E11) in ligand binding to sperm whale myoglobin. Site-directed mutagenesis of a synthetic gene.

1990 ◽  
Vol 265 (20) ◽  
pp. 11788-11795
Author(s):  
K D Egeberg ◽  
B A Springer ◽  
S G Sligar ◽  
T E Carver ◽  
R J Rohlfs ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Sperm Whale ◽  
Site Directed Mutagenesis ◽  
Synthetic Gene ◽  
Directed Mutagenesis ◽  
Sperm Whale Myoglobin

scholarly journals Dual Role of Integrin Alpha-6 in Glioblastoma: Supporting Stemness in Proneural Stem-Like Cells While Inducing Radioresistance in Mesenchymal Stem-Like Cells

Cancers ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 3055
Author(s):  
Elisabetta Stanzani ◽  
Leire Pedrosa ◽  
Guillaume Bourmeau ◽  
Oceane Anezo ◽  
Aleix Noguera-Castells ◽  
...  
Keyword(s):  
Cellular Heterogeneity ◽  
In Vitro Assays ◽  
Rna Seq ◽  
Promising Target ◽  
Damage Response ◽  
Integrin Alpha 6 ◽  
And Control ◽  
Improve Patient

Therapeutic resistance after multimodal therapy is the most relevant cause of glioblastoma (GBM) recurrence. Extensive cellular heterogeneity, mainly driven by the presence of GBM stem-like cells (GSCs), strongly correlates with patients’ prognosis and limited response to therapies. Defining the mechanisms that drive stemness and control responsiveness to therapy in a GSC-specific manner is therefore essential. Here we investigated the role of integrin a6 (ITGA6) in controlling stemness and resistance to radiotherapy in proneural and mesenchymal GSCs subtypes. Using cell sorting, gene silencing, RNA-Seq, and in vitro assays, we verified that ITGA6 expression seems crucial for proliferation and stemness of proneural GSCs, while it appears not to be relevant in mesenchymal GSCs under basal conditions. However, when challenged with a fractionated protocol of radiation therapy, comparable to that used in the clinical setting, mesenchymal GSCs were dependent on integrin a6 for survival. Specifically, GSCs with reduced levels of ITGA6 displayed a clear reduction of DNA damage response and perturbation of cell cycle pathways. These data indicate that ITGA6 inhibition is able to overcome the radioresistance of mesenchymal GSCs, while it reduces proliferation and stemness in proneural GSCs. Therefore, integrin a6 controls crucial characteristics across GBM subtypes in GBM heterogeneous biology and thus may represent a promising target to improve patient outcomes.

scholarly journals The role of amino acid α38 in the control of oxygen binding to human adult and embryonic haemoglobin Portland

1999 ◽  
Vol 343 (3) ◽  
pp. 681-685 ◽  
Author(s):  
Tao ZHENG ◽  
Thomas BRITTAIN ◽  
Nicholas J. WATMOUGH ◽  
Roy E. WEBER
Keyword(s):  
Amino Acid ◽  
Ligand Binding ◽  
Significant Contribution ◽  
Molecular Model ◽  
Site Directed Mutagenesis ◽  
Oxygen Binding ◽  
Naturally Occurring ◽  
Two State Model ◽  
T State

The role of the amino acid at position α38 in haemoglobin has been probed using site-directed mutagenesis. When the Thr residue at position α38 (which is totally conserved in all mammals) is changed to a Gln, the equilibrium properties of the protein are significantly altered. Equilibrium and kinetic data show that the R-state properties of the protein are essentially unaffected by the mutation whilst the allosteric equilibrium and T-state properties are changed. Mutation of the naturally occurring Gln38 of the human embryonic haemoglobin ζ-chain (the only known non-Thr containing globin) to a Thr residue shows the converse change in properties produced by the adult mutation, although in this case the situation is complicated by significant chain heterogeneity in the T state. An extension of the two-state model of co-operativity is presented to describe quantitatively the equilibrium ligand binding in the presence of T-state chain heterogeneity. A molecular model is described in which the putative interaction of αGln38 and βTyr145 is identified which make a significant contribution to the previously reported unusual ligand-binding properties of the ζ-chain containing human embryonic haemoglobins.

scholarly journals Bisphenol A Altersβ-hCG and MIF Release by Human Placenta: AnIn VitroStudy to Understand the Role of Endometrial Cells

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
C. Mannelli ◽  
F. Ietta ◽  
C. Carotenuto ◽  
R. Romagnoli ◽  
A. Z. Szostek ◽  
...  
Keyword(s):  
Bisphenol A ◽  
Conditioned Medium ◽  
Human Placenta ◽  
Environmental Chemicals ◽  
Endometrial Cells ◽  
Direct Exposure ◽  
And Control ◽  
Cell Conditioned Medium

A proper fetomaternal immune-endocrine cross-talk in pregnancy is fundamental for reproductive success. This might be unbalanced by exposure to environmental chemicals, such as bisphenol A (BPA). As fetoplacental contamination with BPA originates from the maternal compartment, this study investigated the role of the endometrium in BPA effects on the placenta. To this end,in vitrodecidualized stromal cells were exposed to BPA 1 nM, and their conditioned medium (diluted 1 : 2) was used on chorionic villous explants from human placenta. Parallel cultures of placental explants were directly exposed to 0.5 nM BPA while, control cultures were exposed to the vehicle (EtOH 0.1%). After 24–48 h, culture medium from BPA-treated and control cultures was assayed for concentration of hormone human Chorionic Gonadotropin (β-hCG) and cytokine Macrophage Migration Inhibitory Factor (MIF). The results showed that direct exposure to BPA stimulated the release of both MIF andβ-hCG. These effects were abolished/diminished in placental cultures exposed to endometrial cell-conditioned medium. GM-MS analysis revealed that endometrial cells retain BPA, thus reducing the availability of this chemical for the placenta. The data obtained highlight the importance ofin vitromodels including the maternal component in reproducing the effects of environmental chemicals on human fetus/placenta.

Diabetes-induced endothelial dysfunction in streptozotocin-treated rats: role of prostaglandin endoperoxides and free radicals.

1993 ◽  
Vol 4 (6) ◽  
pp. 1327-1336
Author(s):  
F X Dai ◽  
A Diederich ◽  
J Skopec ◽  
D Diederich
Keyword(s):  
Free Radicals ◽  
Endothelial Dysfunction ◽  
Diabetic Rats ◽  
Renal Arteries ◽  
Radical Scavenger ◽  
Wistar Kyoto ◽  
And Control ◽  
A2 Receptors

The vasoactive responses of renal arteries from diabetic and control rats were compared in vitro in arteriograph assemblies. Diabetes was established by an iv injection of streptozotocin (55 mg/kg) in Wistar-Kyoto rats. Endothelium-dependent relaxations mediated by nitric oxide (EDNO) were impaired in arteries from the diabetic rats; the impairment in endothelial function increased with duration of the diabetic state. After 6 and 16 wk, the concentrations of acetylcholine required to produce 50% relaxation of norepinephrine preconstriction were 3.2 and 25 microM for arteries from diabetic rats and 0.4 microM in control arteries, representing 8- and 62-fold decreases in sensitivity to the endothelium-dependent vasodilator in the diabetic arteries. After 6 wk of diabetes, renal arteries also became 20-fold less sensitive to relaxation induced by histamine, another agonist that induces EDNO-mediated relaxations. The inhibition of EDNO production with L-NG-nitroarginine produced greater impairments in acetylcholine relaxations in arteries from diabetic rats than from control rats. Relaxations in response to acetylcholine were impaired in arteries from diabetic rats because of increased production of factors that opposed the vasorelaxant effects of EDNO, rather than from decreased production of EDNO. Pretreatment of the diabetic arteries with the hydroxyl radical scavenger dimethylthiourea normalized relaxations in response to acetylcholine. The blockade of prostaglandin H2-thromboxane A2 receptors with SQ 29548 also improved relaxations in response to acetylcholine in diabetic arteries. These data indicate that endothelial dysfunction in the renal arteries of diabetic rats may be mediated by the increased production of free radicals and of prostaglandin endoperoxides, which oppose the vasorelaxant effects of EDNO.

scholarly journals Hemoglobin-spectrin complexes: interference with spectrin tetramer assembly as a mechanism for compartmentalization of band 1 and band 2 complexes

Blood ◽  
1995 ◽  
Vol 86 (1) ◽  
pp. 366-371 ◽  
Author(s):  
CR Kiefer ◽  
JF Trainor ◽  
JB McKenney ◽  
CR Valeri ◽  
LM Snyder
Keyword(s):  
Blood Cell ◽  
Western Blot ◽  
Red Blood Cell ◽  
Blot Analysis ◽  
Natural Phenomenon ◽  
Cell Aging ◽  
Cell Deformability ◽  
Self Association

The irreducible complexation of hemoglobin with spectrin is a natural phenomenon of red blood cell aging, positively correlating with increasing cell density and decreasing cell deformability. The current study begins to address the role of these complexes in the disruption of membrane skeletal physiology and structure. The effect of bound hemoglobin on spectrin dimer self-association was investigated in vitro. The extent of conversion of isolated spectrin dimers to tetramers was evaluated as a function of peroxide-induced globin complexation before the conversion incubations. The incremental accumulation of tetramer was observed to decrease with increasing peroxide concentration used in the globin complexation step. The role of oxidized heme in this process was made apparent by the inability of carboxyhemoglobin to inhibit tetramer accumulation. A Western blot analysis of naturally formed globin-spectrin conjugates demonstrated irreducible complexes of globin with both bands 1 and 2. The complexes are tentatively designated “h1” and “h2”. This analysis also demonstrated that h1 is completely extractable from cell ghosts, whereas h2 is only 50% extractable. These findings are incorporated into a hypothesis linking globin-spectrin complexation and the consequent inhibition of spectrin dimer self-association to the clustered band 3 senescence antigen (Low et al, Science 227:531, 1985).

scholarly journals DSS1 and ssDNA regulate oligomerization of BRCA2

2020 ◽  
Vol 48 (14) ◽  
pp. 7818-7833 ◽  
Author(s):  
Hang Phuong Le ◽  
Xiaoyan Ma ◽  
Jorge Vaquero ◽  
Megan Brinkmeyer ◽  
Fei Guo ◽  
...  
Keyword(s):  
Replication Fork ◽  
Microscopic Analysis ◽  
Filament Formation ◽  
Electron Microscopic ◽  
Microscopic Imaging ◽  
Cellular Events ◽  
Self Association ◽  
Oligomeric Forms

Abstract The tumor suppressor BRCA2 plays a key role in initiating homologous recombination by facilitating RAD51 filament formation on single-stranded DNA. The small acidic protein DSS1 is a crucial partner to BRCA2 in this process. In vitro and in cells (1,2), BRCA2 associates into oligomeric complexes besides also existing as monomers. A dimeric structure was further characterized by electron microscopic analysis (3), but the functional significance of the different BRCA2 assemblies remains to be determined. Here, we used biochemistry and electron microscopic imaging to demonstrate that the multimerization of BRCA2 is counteracted by DSS1 and ssDNA. When validating the findings, we identified three self-interacting regions and two types of self-association, the N-to-C terminal and the N-to-N terminal interactions. The N-to-C terminal self-interaction of BRCA2 is sensitive to DSS1 and ssDNA. The N-to-N terminal self-interaction is modulated by ssDNA. Our results define a novel role of DSS1 to regulate BRCA2 in an RPA-independent fashion. Since DSS1 is required for BRCA2 function in recombination, we speculate that the monomeric and oligomeric forms of BRCA2 might be active for different cellular events in recombinational DNA repair and replication fork stabilization.

scholarly journals Comparative In Vitro and In Silico Analysis of the Selectivity of Indirubin as a Human Ah Receptor Agonist

2018 ◽  
Vol 19 (9) ◽  
pp. 2692 ◽  
Author(s):  
Samantha Faber ◽  
Anatoly Soshilov ◽  
Sara Giani Tagliabue ◽  
Laura Bonati ◽  
Michael Denison
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Ligand Binding ◽  
Species Differences ◽  
In Silico Analysis ◽  
Site Directed Mutagenesis ◽  
Ah Receptor ◽  
Aryl Hydrocarbon ◽  
Silico Analysis

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that modulates gene expression following its binding and activation by structurally diverse chemicals. Species differences in AhR functionality have been observed, with the mouse AhR (mAhR) and human AhR (hAhR) exhibiting significant differences in ligand binding, coactivator recruitment, gene expression and response. While the AhR agonist indirubin (IR) is a more potent activator of hAhR-dependent gene expression than the prototypical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), it is a significantly less potent activator of the mAhR. DNA binding analysis confirmed the greater potency/efficacy of IR in stimulating transformation/DNA binding of the hAhR in vitro and domain-swapping experiments demonstrated that the enhanced response to IR was primarily due to the hAhR ligand binding domain (LBD). Site-directed mutagenesis and functional analysis studies revealed that mutation of H326 and A349 in the mAhR LBD to the corresponding residues in the hAhR LBD significantly increased the potency of IR. Since these mutations had no significant effect on ligand binding, these residues likely contribute to an enhanced efficiency of transformation/DNA binding by IR-bound hAhR. Molecular docking to mAhR LBD homology models further elucidated the different roles of the A375V mutation in TCDD and IR binding, as revealed by [3H]TCDD competitive binding results. These results demonstrate the differential binding of structurally diverse ligands within the LBD of a given AhR and confirm that amino acid differences within the LBD of AhRs contribute to significant species differences in ligand response.

ROLE OF PROTEOLYSIS AT ARGININE-275 OF TISSUE PLASMINOGEN ACTIVATOR (t-PA) AS ASSESSED BY SITE-DIRECTED MUTAGENESIS

1987 ◽  
Author(s):  
G A Vehar ◽  
K M Tate ◽  
D L Higgins ◽  
W E Holmes ◽  
H L Heyneker
Keyword(s):  
Cho Cells ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Single Chain ◽  
Serine Protease Family ◽  
The One ◽  
Chain Form

The significance of the cleavage at arginine-275 of human t-PA has been the subject of debate. It has been reported, as expected for a member of the serine protease family, that the single chain form is a zymogen and that generation of catalytic activity is dependent upon cleavage at arginine-275. Other groups, in contrast, have found considerable enzyme activity associated with the one-chain form of t-PA. To clarify the functional significance of this proteolysis and circumvent cleavage of one-chain t-PA by itself or plasmin, site-directed mutagenesis was employed to change the codon of arginine-275 to specify a glutamic acid. The resulting plasmid was used to transfect CHO cells. The single chain mutant [Glu-275 t-PA] was expressed in CHO cells and the protein purified by conventional techniques. The mutant enzyme could be converted to the two-chain form by V8 protease, but not by plasmin. Glu-275 t-PA was 8 times less active in the cleavage of a tripeptide substrate and 20-50 times less active in the activation of plasminogen in the absence of firbrin(ogen) than its two-chain form. In the presence of fibrin(ogen), in contrast, the one and two-chain forms of Glu-275 t-PA were equal in their ability to activate plasminogen in the presence of fibrin(ogen). The activity in these assays was equal to the activity of wild type t-PA. In addition, it was observed that fibrin bound considerably more of the one-chain form of t-PA than the two chain forms of t-PA and the Glu-275 mutant. The one and two-chain forms of the wild type and mutated t-PA were found to slowly form complexes with plasma protease inhibitors in vitro, although the one-chain forms were less reactive with alpha-2-macroglobulin. It can be concluded that the one-chain form of t-PA appears to be fully functional under physiologic conditions and has an increased affinity for fibrin compared to two-chain t-PA.

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