Function of NoxA1ds in Colon Cancer

2018-08-18

Introduction 

NoxA1ds is derived from a peptide whose structure is based on a short sequence of an essential Nox subunit. It binds directly to NOX1 and displaces NOXA1 to inhibit enzymatic activity and biological function. NoxA1ds is a specific, efficacious, and cell-permeant peptidic inhibitor of Nox1 oxidase. NoxA1ds holds significant promise as a broadly useful inhibitor for testing the functional involvement of Nox1 in myriad pathologies in vitro and in vivo. Moreover, NoxA1ds could be useful as an anti-Nox1 therapeutic to ameliorate disease in its own right or as a peptidomimetic.

Pharmacologic action

NoxA1ds was applied to prepare membrane fractions before addition of fractions containing cytosolic subunits (absent in Nox4 and Nox5 preparations) to maximize its ability to inhibit the oxidase. NoxA1ds did not inhibit Nox2-derived O2- production, Nox4-derived H2O2 production, or Nox5-derived O2- production. When NoxA1ds was added to cell-free preparations of the canonical Nox1 oxidase, composed of catalytic subunit Nox1, activating subunit NOXA1 and organizing subunit NOXO1 along with Rac, NoxA1ds inhibited Nox1-derived O2- production with an IC 50 of 19 nM achieving maximum inhibition of 90% at 1.0 M. NoxA1ds does not scavenge either O2- or H2O2 and does not inhibit Nox2, Nox4, Nox5, or XO activity. NoxA1ds was treated as an isoform-specific inhibitor of Nox1. Membrane-integrated fractions from COS-Nox1 cells containing holoprotein Nox1 with its C-terminal tail were incubated with cumulative concentrations of NoxA1ds (10-12-10-5 M) before adding cytosolic fractions containing NOXA1 and NOXO1. NoxA1ds concentration-dependently inhibited O2- production with an IC50 of 20 nM. Maximal inhibition of Nox1 was achieved at 1.0 M NoxA1ds.

Function

The ability of NoxA1ds to cross the plasma membrane was tested by confocal microscopy in a human colon cancer cell line exclusively expressing Nox1 (HT-29) using FITC-labeled NoxA1ds. As the result showed, NoxA1ds significantly inhibited whole HT-29 carcinoma cell-derivedO2- generation. HT-29 cells were treated with a FITC-labeled NoxA1ds variant for 1 h before imaging. Confocal microscopy revealed that NoxA1ds permeated the cell membrane of HT-29 cells and localized to the cytoplasm. The peptide NoxA1ds is a specific inhibitor of Nox1 and this inhibition occurs via binding to the catalytic Nox1 subunit and blockade of Nox1-NOXA1 binding. NoxA1ds selectively inhibits Nox1-derived O2- production by binding Nox1 and preventing the association of Nox1 with NOXA1. NoxA1ds could potentially mimic functional sites in other proteins and thus interfere with their function. Blast was used to compare the sequence of NoxA1ds to the National Institutes of Health translated nucleotide database to determine potential nonspecific protein interactions with NoxA1ds.

References:

1. Martha Sanchez-Rodriguez1, Mariano Zacarias-Flores2, Alicia Arronte-Rosales1, and Víctor Manuel Mendoza-Nuñez. Antioxidant Effect of Hormone Therapy On Oxidative Stress and Insomnia in Posmenopausal Women. Free Radical Bio. Med. 2011 , 51 (11) :S95-S96.

2. Daniel J. Ranayhossaini, Andres I. Rodriguez, Sanghamitra Sahoo, Beibei B. Chen, Rama K. Mallampalli, Eric E. Kelley, Gabor Csanyi, Mark T. Gladwin, Guillermo Romero, and Patrick J. Pagano. Selective Recapitulation of Conserved and Nonconserved Regions of Putative NOXA1 Protein Activation Domain Confers Isoform-specific Inhibition of Nox1 Oxidase and Attenuation of Endothelial Cell Migration. J. Biological Chem. 2013, 288: 36437-36450.

Creative Peptides has accumulated a huge library of peptide knowledge including frontier peptide articles, application of peptides, useful tools, and more!

 Corticotropin-releasing factor (CRF) is a 41 amino acid peptide that is an important hormone in the hypothalamic ...

 Myristoyl hexapeptide-4, a stabilized peptide, is a synthetic peptide containing lysine, threonine and serine re ...

 The serine/threonine kinase Pim-1 plays an important role in cell cycle progression and apoptosis inhibition, re ...

 MEN 10376 (Asp-Tyr-D-Trp-Val-D-Trp-D-Trp-Lys-NH2) is an analogue of Neurokinin A (NKA), which has a selective af ...

The mammalian precursor gene proglucagon, which contains the glucagon sequence together with two structurally related glucago ...

Contact Us

USA

Address:

Tel: |

Email:

Germany

Address:

Copyright © 2024 Creative Peptides. All rights reserved.