The molecular circadian clock is based on a transcriptional/translational feedback loop in which the stability and half-life of circadian proteins is of importance. Cysteine residues of proteins are subject to several redox reactions leading to S-thiolation and disulfide bond formation, altering protein stability and function. In this work, the ability of the circadian protein period 2 (PER2) to undergo oxidation of cysteine thiols was investigated in HEK-293T cells. PER2 includes accessible cysteines susceptible to oxidation by nitroso cysteine (CysNO), altering its stability by decreasing its monomer form and subsequently increasing PER2 homodimers and multimers. These changes were reversed by treatment with 2-mercaptoethanol and partially mimicked by hydrogen peroxide. These results suggest that cysteine oxidation can prompt PER2 homodimer and multimer formation in vitro, likely by S-nitrosation and disulphide bond formation. These kinds of post-translational modifications of PER2 could be part of the redox regulation of the molecular circadian clock.

https://www.mdpi.com/2218-273X/12/7/892/pdf?version=1656304238

Cysteine Oxidation Promotes Dimerization/Oligomerization of Circadian Protein Period 2

Precise control of pluripotency is a requirement for the safe and effective use of hPSCs in research and therapies. Here we report that pyruvate dehydrogenase upregulates histone H3 pan acetylation and levels of pluripotency marker NANOG in 5% O2. Pyruvate dehydrogenase (PDH) is an essential metabolic switch and a bottleneck for the glycolytic production of acetyl-CoA. Silencing of gene expression showed that PDH is regulated by the activity of its phosphatase PDP1. We show that PDP1 is sensitive to reactive oxygen species-mediated inactivation, leading to the downregulation of H3 pan acetylation and NANOG levels. Furthermore, we show that FGF2, a cytokine commonly used to maintain pluripotency activates pyruvate dehydrogenase through MEK1/2-ERK1/2 signaling pathway-mediated downregulation of ROS in 5% O2, thus promoting histone acetylation. Our results show the importance of pyruvate dehydrogenase in regulating energy metabolism and its connection to pluripotency. Furthermore, our data highlight the role of reactive oxygen species and redox homeostasis in pluripotency maintenance and differentiation. Highlights - PDP1-induced activation of PDH leads to increased histone H3 pan acetylation and NANOG levels in hPSCs - Reactive oxygen species (ROS) inactivate PDP1 and decrease histone H3 pan acetylation and NANOG levels in hPSCs - MEK1/2-ERK1/2 signaling-mediated downregulation of ROS in 5% O2 activates PDH in hPSCs Graphical abstract

FGF2-induced Redox Signaling: A Mechanism Regulating Pyruvate Dehydrogenase Driven Histone Acetylation and NANOG Upregulation

Allantopyrone A interferes with the degradation of hypoxia-inducible factor 1α protein by reducing proteasome activity in human fibrosarcoma HT-1080 cells

Cancer cells, when exposed to the hypoxic tumour microenvironment, respond by activating hypoxia‐inducible factors (HIFs). HIF‐1 mediates extensive metabolic re‐programming, and expression of HIF‐1α, its oxygen‐regulated subunit, is associated with poor prognosis in cancer. Here we analyse the role of pyruvate dehydrogenase phosphatase 1 (PDP1) in the regulation of HIF‐1 activity. PDP1 is a key hormone‐regulated metabolic enzyme that dephosphorylates and activates pyruvate dehydrogenase (PDH), thereby stimulating the conversion of pyruvate into acetyl‐CoA. Silencing of PDP1 down‐regulated HIF transcriptional activity and the expression of HIF‐dependent genes, including that of PDK1, the kinase that phosphorylates and inactivates PDH, opposing the effects of PDP1. Inversely, PDP1 stimulation enhanced HIF activity under hypoxia. Alteration of PDP1 levels or activity did not have an effect on HIF‐1α protein levels, nuclear accumulation or interaction with its partners ARNT and NPM1. However, depletion of PDP‐1 decreased histone H3 acetylation of HIF‐1 target gene promoters and inhibited binding of HIF‐1 to the respective hypoxia‐response elements (HREs) under hypoxia. Furthermore, the decrease of HIF transcriptional activity upon PDP1 depletion could be reversed by treating the cells with acetate, as an exogenous source of acetyl‐CoA, or the histone deacetylase (HDAC) inhibitor trichostatin A. These data suggest that the PDP1/PDH/HIF‐1/PDK1 axis is part of a homeostatic loop which, under hypoxia, preserves cellular acetyl‐CoA production to a level sufficient to sustain chromatin acetylation and transcription of hypoxia‐inducible genes.

Pyruvate dehydrogenase phosphatase 1 (PDP1) stimulates HIF activity by supporting histone acetylation under hypoxia

The study of the circadian clock and its mechanisms is easily facilitated through clock resetting in cell culture. Among the various established synchronizers of the circadian clock in cell culture (temperature, serum shock, glucocorticoids), the artificial glucocorticoid Dexamethasone (DEX) is the most widely used. DEX treatment as a protocol to reset the circadian clock in culture gives simple readout with minimal laboratory requirements. Even though there are many studies regarding clock resetting in culture using DEX, reference points or expression patterns of core clock genes and their protein products are scarce and sometimes contradict other works with similar methodology. We synchronise a cell line of human origin with DEX to be used for studies on circadian rhythms.We treat HEK 293T cells with DEX and describe the patterns of mRNA and proteins of core clock regulators, while making a clear point on how CLOCK is less than an ideal molecule to help monitor rhythms in this cell line. 

https://bmcresnotes.biomedcentral.com/counter/pdf/10.1186/s13104-021-05871-7

Core clock regulators in dexamethasone-treated HEK 293T cells at 4 h intervals

/pdf/core-clock-regulators-in-dexamethasone-treated-hek-293t-ioi9uu1o.pdf

Amalia Kanoura

Papers

Pyruvate dehydrogenase phosphatase 1 (PDP1) stimulates HIF activity by supporting histone acetylation under hypoxia

Core clock regulators in dexamethasone-treated HEK 293T cells at 4 h intervals

Core clock regulators in dexamethasone-treated HEK 293T cells at 4 h intervals