Monoclonal Antibody-Based Screening Assay for Factor Inhibiting Hypoxia-Inducible Factor Inhibitors

2008 ◽  
Vol 13 (6) ◽  
pp. 494-503 ◽  
Author(s):  
Sang-Hyeup Lee ◽  
Jeong Hee Moon ◽  
Eun Ah Cho ◽  
Seong-Eon Ryu ◽  
Myung Kyu Lee
Keyword(s):  
Monoclonal Antibody ◽  
High Throughput Screening ◽  
Transcriptional Activity ◽  
Hypoxia Inducible Factor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sensitive Assay ◽  
Screening Assay ◽  
Hypoxic Conditions

The factor-inhibiting hypoxia-inducible factor (FIH) hydroxylates the asparagine 803 (Asn803) residue of the hypoxia-inducible factor 1α (HIF-1α), and the modification abrogates the transcriptional activity of HIF-1α. Because FIH is more active on HIF-1α than prolyl hydroxylase domain proteins under hypoxic conditions, its inhibitors have potential to be developed as anti-ischemic drugs targeting normal cells stressed by hypoxia. In this study, the authors developed the first monoclonal antibody, SHN-HIF1α, specifically to Asn803 hydroxylated HIF-1α and a sensitive assay system for FIH inhibitors using the monoclonal antibody (Mab). SHN-HIF1α showed 740 times higher affinity to the Asn803 hydroxylated HIF-1α peptide than the unmodified one. An enzyme-linked immunosorbent assay—based system using SHN-HIF1α displayed at least 30 times more sensitivity than previous methods for screening FIH inhibitors and was easily applicable to develop a high-throughput screening system. SHN-HIF1α also showed an Asn803 hydroxylation-dependent specificity to HIF-1α in cells. Taken together, the results suggest that it may be used to analyze the in vivo and in vitro activities of FIH inhibitors. ( Journal of Biomolecular Screening 2008:494-503)

scholarly journals A Novel Mouse Monoclonal Antibody C42 against C-Terminal Peptide of Alpha-1-Antitrypsin

2021 ◽  
Vol 22 (4) ◽  
pp. 2141
Author(s):  
Srinu Tumpara ◽  
Elena Korenbaum ◽  
Mark Kühnel ◽  
Danny Jonigk ◽  
Beata Olejnicka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Western Blotting ◽  
Complex Mixture ◽  
Immunoglobulin M ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359–394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1–0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.

scholarly journals USP29-mediated HIF1α stabilization is associated with Sorafenib resistance of hepatocellular carcinoma cells by upregulating glycolysis

Oncogenesis ◽  
2021 ◽  
Vol 10 (7) ◽  
Author(s):  
Ruize Gao ◽  
David Buechel ◽  
Ravi K. R. Kalathur ◽  
Marco F. Morini ◽  
Mairene Coto-Llerena ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Transcriptional Activity ◽  
Kinase Inhibitor ◽  
Aerobic Glycolysis ◽  
Hypoxia Inducible Factor ◽  
Aberrant Expression ◽  
Key Enzyme ◽  
Hcc Cells

AbstractUnderstanding the mechanisms underlying evasive resistance in cancer is an unmet medical need to improve the efficacy of current therapies. In hepatocellular carcinoma (HCC), aberrant expression of hypoxia-inducible factor 1 α (HIF1α) and increased aerobic glycolysis metabolism are drivers of resistance to therapy with the multi-kinase inhibitor Sorafenib. However, it has remained unknown how HIF1α is activated and how its activity and the subsequent induction of aerobic glycolysis promote Sorafenib resistance in HCC. Here, we report the ubiquitin-specific peptidase USP29 as a new regulator of HIF1α and of aerobic glycolysis during the development of Sorafenib resistance in HCC. In particular, we identified USP29 as a critical deubiquitylase (DUB) of HIF1α, which directly deubiquitylates and stabilizes HIF1α and, thus, promotes its transcriptional activity. Among the transcriptional targets of HIF1α is the gene encoding hexokinase 2 (HK2), a key enzyme of the glycolytic pathway. The absence of USP29, and thus of HIF1α transcriptional activity, reduces the levels of aerobic glycolysis and restores sensitivity to Sorafenib in Sorafenib-resistant HCC cells in vitro and in xenograft transplantation mouse models in vivo. Notably, the absence of USP29 and high HK2 expression levels correlate with the response of HCC patients to Sorafenib therapy. Together, the data demonstrate that, as a DUB of HIF1α, USP29 promotes Sorafenib resistance in HCC cells, in parts by upregulating glycolysis, thereby opening new avenues for therapeutically targeting Sorafenib-resistant HCC in patients.

Activating transcription factor 2 increases transactivation and protein stability of hypoxia-inducible factor 1α in hepatocytes

2009 ◽  
Vol 424 (2) ◽  
pp. 285-296 ◽  
Author(s):  
Jeong Hae Choi ◽  
Hyun Kook Cho ◽  
Yung Hyun Choi ◽  
JaeHun Cheong
Keyword(s):  
Transcription Factor ◽  
Protein Stability ◽  
Gene Transcription ◽  
Hypoxia Inducible Factor ◽  
Protein Stabilization ◽  
Hypoxic Conditions ◽  
Tumour Suppressor Protein ◽  
Activating Transcription Factor

HIF-1 (hypoxia inducible factor 1) performs a crucial role in mediating the response to hypoxia. However, other transcription factors are also capable of regulating hypoxia-induced target-gene transcription. In a previous report, we demonstrated that the transcription factor ATF-2 (activating transcription factor 2) regulates hypoxia-induced gene transcription, along with HIF-1α. In the present study, we show that the protein stability of ATF-2 is induced by hypoxia and the hypoxia-mimic CoCl2 (cobalt chloride), and that ATF-2 induction enhances HIF-1α protein stability via direct protein interaction. The knockdown of ATF-2 using small interfering RNA and translation-inhibition experiments demonstrated that ATF-2 plays a key role in the maintenance of the expression level and transcriptional activity of HIF-1α. Furthermore, we determined that ATF-2 interacts directly with HIF-1α both in vivo and in vitro and competes with the tumour suppressor protein p53 for HIF-1α binding. Collectively, these results show that protein stabilization of ATF-2 under hypoxic conditions is required for the induction of the protein stability and transactivation activity of HIF-1α for efficient hypoxia-associated gene expression.

Hypoxia induces B-type natriuretic peptide release in cell lines derived from human cardiomyocytes

2009 ◽  
Vol 297 (2) ◽  
pp. H550-H555 ◽  
Author(s):  
Gregori Casals ◽  
Josefa Ros ◽  
Alessandro Sionis ◽  
Mercy M. Davidson ◽  
Manuel Morales-Ruiz ◽  
...  
Keyword(s):  
Natriuretic Peptide ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Ventricular Myocytes ◽  
Hypoxia Inducible Factor ◽  
Peptide Hormone ◽  
Mrna Levels ◽  
Human Cardiomyocytes ◽  
Hypoxic Conditions

B-type natriuretic peptide (BNP) is a peptide hormone of myocardial origin with significant cardioprotective properties. Patients with myocardial ischemia present with high levels of BNP in plasma and elevated expression in the myocardium. However, the molecular mechanisms of BNP induction in the ischemic myocardium are not well understood. The aim of the investigation was to assess whether myocardial hypoxia induces the production of BNP in human ventricular myocytes. To test the hypothesis that reduced oxygen tension can directly stimulate BNP gene expression and release in the absence of hemodynamic or neurohormonal stimuli, we used an in vitro model system of cultured human ventricular myocytes (AC16 cells). Cells were cultured under normoxic (21% O2) or hypoxic (5% O2) conditions for up to 48 h. The accumulation of BNP, atrial natriuretic peptide (ANP), and vascular endothelial growth factor (VEGF) was then measured. Hypoxia stimulated the protein release of BNP and VEGF but not ANP. In concordance, the increased mRNA levels of BNP and VEGF but not ANP were found on culturing AC16 cells under hypoxic conditions. The analysis of the transcriptional activity of the hypoxia-inducible factor 1 (HIF-1) in nuclear extracts showed that HIF-1 activity was induced under hypoxic conditions. Finally, the treatment of AC16 cells with the HIF-1 inhibitor rotenone in hypoxia inhibited BNP and VEGF release. In conclusion, these data indicate that hypoxia induces the synthesis and secretion of BNP in human ventricular myocytes, likely through HIF-1-enhanced transcriptional activity.

Abstract 34: Statins Directly Activate Erk5 and Reduce Endothelial Dysfunction and Acute Allograft Rejection

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Yuichiro Takei ◽  
Nhat-Tu Le ◽  
Hakjoo Lee ◽  
Kyung-Sun Heo ◽  
Cheryl Hurley ◽  
...  
Keyword(s):  
Allograft Rejection ◽  
High Throughput Screening ◽  
Transcriptional Activity ◽  
Vascular Reactivity ◽  
Pleiotropic Effects ◽  
Protective Effects ◽  
Acute Allograft Rejection

Rationale: The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, or statins, are principal therapeutic agents for the treatment of hypercholesteremia. Statins, however, appear to also exert cholesterol-independent pleiotropic effects, such as improvement of endothelial (EC) function, stabilization of fibrous plaques, and decrease vascular inflammation. It is now well established that statins are beneficial in the prevention of cardiovascular disease and also widely used for suppressing cardiac allograft rejection. Previously, we have reported anti-inflammatory effect of ERK5 kinase in ECs. Methods and Results: In this study, we screened small molecules that activate ERK5 using high throughput screening, and identified statins as strong activators of the transcriptional activity of ERK5. In particular, we have found that pitavastatin increases ERK5 transcriptional activity, KLF2 promoter activity, and eNOS mRNA expression in ECs. These effects are abolished by the depletion of ERK5, but not its direct upstream kinase, MEK5. In addition, pitavastatin directly and dose-dependently activates ERK5 kinase activity in an in vitro kinase reaction assay, suggesting that ERK5 is a direct target of this statin. To examine the functional role of EC ERK5 activation by the statin in vivo, we utilized inducible endothelial ERK5 knock out (EC-ERK5-KO) mice and evaluated the effect of pitavastatin on EC function and acute allograft rejection. Depletion of ERK5 in ECs resulted in significant EC inflammation and dysfunction in vivo. Although pitavastatin reduced leukocyte rolling and vascular reactivity in mesebteruc microvessels of diabetic mice and prolonged allograft survival in a full allomismatch combination model, these protective effects were lost in EC-ERK5-KO mice. Conclusion: These data suggest the crucial role of ERK5 in pleiotropic effects of statins on EC dysfunction and allograft rejection in vivo.

scholarly journals Characterization of novel neutralizing mouse monoclonal antibody JM1-24-3 developed against MUC18 in metastatic melanoma

2020 ◽  
Vol 39 (1) ◽  
Author(s):  
Runhua Feng ◽  
Yuling Wang ◽  
Vijaya Ramachandran ◽  
Qinhong Ma ◽  
Matthew M. May ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Metastatic Melanoma ◽  
Therapeutic Target ◽  
High Throughput Screening ◽  
Conformational Epitope ◽  
Migration And Invasion ◽  
Melanoma Tumor ◽  
Downstream Signaling

Abstract Background MUC18 is a glycoprotein highly expressed on the surface of melanoma and other cancers which promotes tumor progression and metastasis. However, its mechanism of action and suitability as a therapeutic target are unknown. Methods A monoclonal antibody (mAb) (JM1-24-3) was generated from metastatic melanoma tumor live cell immunization, and high-throughput screening identified MUC18 as the target. Results Analysis of molecular interactions between MUC18 and JM1-24-3 revealed that the downstream signaling events depended on binding of the mAb to a conformational epitope on the extracellular domain of MUC18. JM1-24-3 inhibited melanoma cell proliferation, migration and invasion in vitro and reduced tumor growth and metastasis in vivo. Conclusion These results confirm that MUC18 is mechanistically important in melanoma growth and metastasis, suggest that the MUC18 epitope identified is a promising therapeutic target, and that the JM1-24-3 mAb may serve as the basis for a potential therapeutic agent.

scholarly journals A Recombinant Human Monoclonal Antibody to Human Metapneumovirus Fusion Protein That Neutralizes Virus In Vitro and Is Effective Therapeutically In Vivo

2007 ◽  
Vol 81 (15) ◽  
pp. 8315-8324 ◽  
Author(s):  
John V. Williams ◽  
Zhifeng Chen ◽  
Gabriella Cseke ◽  
David W. Wright ◽  
Christopher J. Keefer ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Human Monoclonal Antibody ◽  
Virus Titer ◽  
Human Metapneumovirus ◽  
Enzyme Linked Immunosorbent Assay ◽  
F Protein ◽  
Fold Reduction ◽  
Hmpv Infection

ABSTRACT Human metapneumovirus (hMPV) is a recently discovered paramyxovirus that is a major cause of lower-respiratory-tract disease. hMPV is associated with more severe disease in infants and persons with underlying medical conditions. Animal studies have shown that the hMPV fusion (F) protein alone is capable of inducing protective immunity. Here, we report the use of phage display technology to generate a fully human monoclonal antibody fragment (Fab) with biological activity against hMPV. Phage antibody libraries prepared from human donor tissues were selected against recombinant hMPV F protein with multiple rounds of panning. Recombinant Fabs then were expressed in bacteria, and supernatants were screened by enzyme-linked immunosorbent assay and immunofluorescent assays. A number of Fabs that bound to hMPV F were isolated, and several of these exhibited neutralizing activity in vitro. Fab DS7 neutralized the parent strain of hMPV with a 60% plaque reduction activity of 1.1 μg/ml and bound to hMPV F with an affinity of 9.8 ×10−10 M, as measured by surface plasmon resonance. To test the in vivo activity of Fab DS7, groups of cotton rats were infected with hMPV and given Fab intranasally 3 days after infection. Nasal turbinates and lungs were harvested on day 4 postinfection and virus titers determined. Animals treated with Fab DS7 exhibited a >1,500-fold reduction in viral titer in the lungs, with a modest 4-fold reduction in the nasal tissues. There was a dose-response relationship between the dose of DS7 and virus titer. Human Fab DS7 may have prophylactic or therapeutic potential against severe hMPV infection.

scholarly journals The Phosphatase SHP-2 Activates HIF-1α in Wounds In Vivo by Inhibition of 26S Proteasome Activity

2019 ◽  
Vol 20 (18) ◽  
pp. 4404 ◽  
Author(s):  
Yvonn Heun ◽  
Katharina Grundler Groterhorst ◽  
Kristin Pogoda ◽  
Bjoern F Kraemer ◽  
Alexander Pfeifer ◽  
...  
Keyword(s):  
Tyrosine Phosphatase ◽  
Hypoxia Inducible Factor ◽  
Proteasome Activity ◽  
26S Proteasome ◽  
Mitogen Activated Protein Kinase ◽  
Hypoxic Conditions ◽  
Mitogen Activated Protein ◽  
Specific Regulation

Vascular remodeling and angiogenesis are required to improve the perfusion of ischemic tissues. The hypoxic environment, induced by ischemia, is a potent stimulus for hypoxia inducible factor 1α (HIF-1α) upregulation and activation, which induce pro-angiogenic gene expression. We previously showed that the tyrosine phosphatase SHP-2 drives hypoxia mediated HIF-1α upregulation via inhibition of the proteasomal pathway, resulting in revascularization of wounds in vivo. However, it is still unknown if SHP-2 mediates HIF-1α upregulation by affecting 26S proteasome activity and how the proteasome is regulated upon hypoxia. Using a reporter construct containing the oxygen-dependent degradation (ODD) domain of HIF-1α and a fluorogenic proteasome substrate in combination with SHP-2 mutant constructs, we show that SHP-2 inhibits the 26S proteasome activity in endothelial cells under hypoxic conditions in vitro via Src kinase/p38 mitogen-activated protein kinase (MAPK) signalling. Moreover, the simultaneous expression of constitutively active SHP-2 (E76A) and inactive SHP-2 (CS) in separate hypoxic wounds in the mice dorsal skin fold chamber by localized magnetic nanoparticle-assisted lentiviral transduction showed specific regulation of proteasome activity in vivo. Thus, we identified a new additional mechanism of SHP-2 mediated HIF-1α upregulation and proteasome activity, being functionally important for revascularization of wounds in vivo. SHP-2 may therefore constitute a potential novel therapeutic target for the induction of angiogenesis in ischemic vascular disease.

Transcriptional regulation mechanisms of hypoxia-induced neuroglobin gene expression

2012 ◽  
Vol 443 (1) ◽  
pp. 153-164 ◽  
Author(s):  
Ning Liu ◽  
Zhanyang Yu ◽  
Shuanglin Xiang ◽  
Song Zhao ◽  
Anna Tjärnlund-Wolf ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hypoxia Inducible Factor ◽  
Gene Promoter ◽  
Nuclear Factor Κb ◽  
Proximal Promoter ◽  
Mobility Shift ◽  
Hypoxic Conditions ◽  
Mobility Shift Assay

Ngb (neuroglobin) has been identified as a novel endogenous neuroprotectant. However, little is known about the regulatory mechanisms of Ngb expression, especially under conditions of hypoxia. In the present study, we located the core proximal promoter of the mouse Ngb gene to a 554 bp segment, which harbours putative conserved NF-κB (nuclear factor κB)- and Egr1 (early growth-response factor 1) -binding sites. Overexpression and knockdown of transcription factors p65, p50, Egr1 or Sp1 (specificity protein 1) increased and decreased Ngb expression respectively. Experimental assessments with transfections of mutational Ngb gene promoter constructs, as well as EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation) assays, demonstrated that NF-κB family members (p65, p50 and cRel), Egr1 and Sp1 bound in vitro and in vivo to the proximal promoter region of the Ngb gene. Moreover, a κB3 site was found as a pivotal cis-element responsible for hypoxia-induced Ngb promoter activity. NF-κB (p65) and Sp1 were also responsible for hypoxia-induced up-regulation of Ngb expression. Although there are no conserved HREs (hypoxia-response elements) in the promoter of the mouse Ngb gene, the results of the present study suggest that HIF-1α (hypoxia-inducible factor-1α) is also involved in hypoxia-induced Ngb up-regulation. In conclusion, we have identified that NF-κB, Egr1 and Sp1 played important roles in the regulation of basal Ngb expression via specific interactions with the mouse Ngb promoter. NF-κB, Sp1 and HIF-1α contributed to the up-regulation of mouse Ngb gene expression under hypoxic conditions.

scholarly journals Diphenylpyrazole-Derived Compounds Increase Survival Time of Mice after Prion Infection

2011 ◽  
Vol 55 (10) ◽  
pp. 4774-4781 ◽  
Author(s):  
Fabienne Leidel ◽  
Martin Eiden ◽  
Markus Geissen ◽  
Hans A. Kretzschmar ◽  
Armin Giese ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Natural Infection ◽  
New Drugs ◽  
Transmissible Spongiform Encephalopathies ◽  
Spongiform Encephalopathy ◽  
Variant Form ◽  
Screening Assay ◽  
High Throughput Screening Assay

ABSTRACTTransmissible spongiform encephalopathies (TSEs) represent a group of fatal neurodegenerative disorders that can be transmitted by natural infection or inoculation. TSEs include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, and Creutzfeldt-Jakob disease (CJD) in humans. The emergence of a variant form of CJD (vCJD), which has been associated with BSE, produced strong pressure to search for effective treatments with new drugs. Up to now, however, TSEs have proved incurable, although many efforts have been made bothin vitroandin vivoto search for potent therapeutic and prophylactic compounds. For this purpose, we analyzed a compound library consisting of 10,000 compounds with a cell-based high-throughput screening assay dealing with scrapie-infected scrapie mouse brain and ScN2A cells and identified a new class of inhibitors consisting of 3,5-diphenylpyrazole (DPP) derivatives. The most effective DPP derivative showed half-maximal inhibition of PrPScformation at concentrations (IC50) of 0.6 and 1.2 μM, respectively. This compound was subsequently subjected to a number of animal experiments using scrapie-infected wild-type C57BL/6 and transgenic Tga20 mice. The DPP derivative induced a significant increase of incubation time both in therapeutic and prophylactic experiments. The onset of the prion disease was delayed by 37 days after intraperitoneal and 42 days after oral application, respectively. In summary, we demonstrate a highin vitroefficiency of DPP derivatives against prion infections that was substantiatedin vivofor one of these compounds. These results indicate that the novel class of DPP compounds should comprise excellent candidates for future therapeutic studies.

Sign in / Sign up

Export Citation Format

Share Document