Hello guys,
I wonder if there is any assay that could be used to determine the redox state of HMGB1 protein released in culture media ? your answers will be greatly appreciated
Dear Sir. Concerning your issue about the assays are used to determine the redox state of HMGB1. During inflammation, high-mobility group box 1 in reduced all-thiol form (at-HMGB1) takes charge of chemoattractant activity, whereas only disulfide-HMGB1 (ds-HMGB1) has cytokine activity. Also as pro-angiogenic inducer, the role of HMGB1 in different redox states has never been defined in tumour angiogenesis. To verify which redox states of HMGB1 induces angiogenesis in colorectal carcinoma. To measure the expression of VEGF-A and angiogenic properties of the endothelial cells (ECs), at-HMGB1 or ds-HMGB1 was added to cell medium, further with their special inhibitors (DPH1.1 mAb and 2G7 mAb) and antibodies of corresponding receptors (RAGE Ab and TLR4 Ab). Also, a co-culture system and conditioned medium from tumour cells were applied to mimic tumour microenvironment. HMGB1 triggered VEGF-A secretion mainly through its disulfide form interacting with TLR4, while co-operation of at-HMGB1 and RAGE mediated migratory capacity of ECs. Functional inhibition of HMGB1 and its receptors abrogated HMGB1-induced angiogenic properties of ECs co-cultured with tumour cells. HMGB1 orchestrates the key events of tumour angiogenesis, migration of ECs and their induction to secrete VEGF-A, by adopting distinct redox states. high-mobility group box protein 1 (HMGB1) is a nonhistone chromatin-binding protein involved in the regulation of transcription. Extracellularly, HMGB1 acts as a danger molecule with properties of a proinflammatory cytokine. It can be actively secreted from myeloid cells or passively leak from any type of injured, necrotic cell. Increased serum levels of active HMGB1 are often found in pathogenic inflammatory conditions and correlate with worse prognoses in cancer, sepsis, and autoimmunity. By damaging cells, superoxide and peroxynitrite promote leakage of HMGB1. Recent Advances: The activity of HMGB1 strongly depends on its redox state: Inflammatory-active HMGB1 requires an intramolecular disulfide bond (Cys23 and Cys45) and a reduced Cys106. Oxidation of the latter blocks its stimulatory activity and promotes immune tolerance. Critical Issues: Reactive oxygen and nitrogen species create an oxidative environment and can be detoxified by superoxide dismutase (SOD), catalase, and peroxidases. Modifications of the oxidative environment influence HMGB1 activity. Future Directions: In this review, we hypothesize that manipulations of an oxidative environment by SOD mimics or by hydrogen sulfide are prone to decrease tissue damage. Both the concomitant decreased HMGB1 release and its redox chemical modifications ameliorate inflammation and tissue damage. I think the following below links may help you in your analysis:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4568917/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3928832/
https://arthritis-research.biomedcentral.com/articles/10.1186/s13075-015-0856-2
Thanks
Thank you Isam for your reply.
In fact if anyone tried to purify released HMGB1 from cell culture media and was successful to check for its redox state (disulfide/reduced/oxidized) using any type of assays if someone did this and want to provide me with more details on how to do it, this would be great.
I know i can purchase HMGB1 protein in any redox form from HMGBbiotech, however what i want to do is to check the redox state of the released HMGB1 myself ?
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