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1.
Biochimie ; 170: 106-117, 2020 Mar.
Article in English | MEDLINE | ID: mdl-31887335

ABSTRACT

Cholesterol biosynthesis is a multistep process in mammals that includes the aerobic removal of three methyl groups from the intermediate lanosterol, one from position 14 and two from position 4. During the demethylations at position 4, a 3-ketosteroid reductase catalyses the conversion of both 4-methylzymosterone and zymosterone to 4-methylzymosterol and zymosterol, respectively, restoring the alcoholic function of lanosterol, which is also maintained in cholesterol. Unlike other eukaryotes, mammals also use the same enzyme as an estrone reductase that can transform estrone (E1) into estradiol (E2). This enzyme, named 17ß-hydroxysteroid dehydrogenase type 7 (HSD17B7), is therefore a multifunctional protein in mammals, and one that belongs to both the HSD17B family, which is involved in steroid-hormone metabolism, and to the family of post-squalene cholesterol biosynthesis enzymes. In the present study, a series of known inhibitors of human HSD17B7's E1-reductase activity have been assayed for potential inhibition against 3-ketosteroid reductase activity. Surprisingly, the assayed compounds lost their inhibition activity when tested in HepG2 cells that were incubated with radiolabelled acetate and against the recombinant overexpressed human enzyme incubated with 4-methylzymosterone (both radiolabelled and not). Preliminary kinetic analyses suggest a mixed or non-competitive inhibition on the E1-reductase activity, which is in agreement with Molecular Dynamics simulations. These results raise questions about the mechanism(s) of action of these possible inhibitors, the enzyme dynamic regulation and the interplay between the two activities.


Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/metabolism , Enzyme Inhibitors/pharmacology , Estradiol/metabolism , Estrone/metabolism , S100 Calcium Binding Protein A6/antagonists & inhibitors , S100 Calcium Binding Protein A6/metabolism , 3-Hydroxysteroid Dehydrogenases/chemistry , 3-Hydroxysteroid Dehydrogenases/metabolism , Cholesterol/metabolism , Enzyme Inhibitors/chemistry , Estrogens/metabolism , Hep G2 Cells , Humans , Protein Conformation
2.
J Cell Physiol ; 234(10): 17561-17569, 2019 08.
Article in English | MEDLINE | ID: mdl-30805941

ABSTRACT

Epidermal growth factor receptor (EGFR) is a central transmitter of mitogenic signals in epithelial cells; enhanced EGFR activity is observed in many tumors of epithelial origin. S100A6 is a small calcium-binding protein, characteristic mainly of epithelial cells and fibroblasts, strongly implicated in cell proliferation and upregulated in tumors. In this study, using biochemical assays along with immunohistochemical and immunocytochemical analysis of organotypic and standard cultures of HaCaT keratinocytes with S100A6 overexpression or knock-down, we have examined the effect of S100A6 on EGFR activity and downstream signaling. We found that HaCaT cells overexpressing S100A6 had enhanced EGFR, phospho EGFR, and phospho extracellular signal-regulated kinase 1/2 (pERK1/2) staining intensity and level coupled to higher signal transducer and activator of transcription 3 (STAT3) activity. Conversely, S100A6 knockdown cells had impaired EGFR signaling that could be enhanced by addition of recombinant S100A6 to the culture media. Altogether the results show that S100A6 may exert its proproliferative effects through activating EGFR.


Subject(s)
Cell Cycle Proteins/metabolism , Keratinocytes/metabolism , S100 Calcium Binding Protein A6/metabolism , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Cell Line , Cell Proliferation/physiology , ErbB Receptors/metabolism , Gene Knockdown Techniques , Humans , Keratinocytes/cytology , MAP Kinase Signaling System , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , S100 Calcium Binding Protein A6/antagonists & inhibitors , S100 Calcium Binding Protein A6/genetics , STAT3 Transcription Factor/metabolism , Signal Transduction , Transforming Growth Factor alpha/metabolism
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