title: Mechanistic insights into the inhibitory effects of palmitoylation on cytosolic thioredoxin reductase and thioredoxin.
abstract: Overnutrition can lead to oxidative stress, but its underlying mechanism remains unclear. In this study, we report that human liver-derived HepG2 cells utilize cytosolic thioredoxin reductase (TrxR1) and thioredoxin (hTrx1) to defend against the high glucose/palmitate-mediated increase in reactive oxygen species. However, enhanced TrxR1/hTrx1 palmitoylation occurs in parallel with a decrease in their activities under the conditions studied here. An autoacylation process appears to be the major mechanism for generating palmitoylated TrxR1/Trx1 in HepG2 cells. A novel feature of this post-translational modification is the covalent inhibition of TrxR1/hTrx1 by palmitoyl-CoA, an activated form of palmitate. The palmitoyl-CoA/TrxR1 reaction is NADPH-dependent and produces palmitoylated TrxR1 at an active site selenocysteine residue. Conversely, S-palmitoylation occurs at the structural Cys62/Cys69/Cys72 residues but not the active site Cys32/Cys35 residues of hTrx1. Palmitoyl-CoA concentration and the period of incubation with TrxR1/hTrx1 are important factors that influence the inhibitory efficacy of palmitoyl-CoA on TrxR1/hTrx1. Thus, an increase in TrxR1/hTrx1 palmitoylation could be a potential consequence of high glucose/palmitate. The time-dependent inactivation of the NADPH-TrxR1-Trx1 system by palmitoyl-CoA occurs in a biphasic manner - a fast phase followed by a slow phase. Kinetic analysis suggests that the fast phase is consistent with a fast and reversible association between TrxR1/hTrx1 and palmitoyl-CoA. The slow phase is correlated with a slow and irreversible inactivation, in which selenolate/thiolate groups nucleophilically attack the α-carbon of bound palmitoyl-CoA, leading to the formation of thioester/selenoester bonds. hTrx1 can enhance rate of fast phase but limits the rate of slow phase when it is present in a preincubation mixture containing NADPH, TrxR1 and palmitoyl-CoA. Therefore, hTrx1 may provide palmitoylation sites or partially protect the TrxR1 active site selenol/thiol group(s) from palmitoylation. Our data suggest that Se/S-palmitoylation acts as an important modulator of TrxR1/hTrx1 activities, representing a novel potential mechanism that underlies overnutrition-induced events.
authors.affiliation: College of Life Sciences, University of Chinese Academy of Sciences, Yuquan Road 19(A), 100049 Beijing, China.
authors.forename: Huijun
authors.initials: H
authors.lastname: Qin
authors.affiliation: College of Life Sciences, University of Chinese Academy of Sciences, Yuquan Road 19(A), 100049 Beijing, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Liang
authors.affiliation: College of Life Sciences, University of Chinese Academy of Sciences, Yuquan Road 19(A), 100049 Beijing, China.
authors.forename: Zhiyu
authors.initials: Z
authors.lastname: Xu
authors.affiliation: Institute of Materia Medica, Chinese Academy of Medical Sciences & Perking Union Medical College, 100050 Beijing, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Ye
authors.affiliation: The State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, 100101 Beijing, China.
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Li
authors.affiliation: College of Life Sciences, University of Chinese Academy of Sciences, Yuquan Road 19(A), 100049 Beijing, China. Electronic address: liazho@ucas.ac.cn.
authors.forename: Liangwei
authors.initials: L
authors.lastname: Zhong
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D002417:Cattle; D003600:Cytosol / Q000201:enzymology*; D005947:Glucose / Q000494:pharmacology; D056945:Hep G2 Cells; D006801:Humans; D054878:Lipoylation / Q000187:drug effects; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction / Q000187:drug effects; D010168:Palmitates / Q000494:pharmacology; D010171:Palmitoyl Coenzyme A / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D013880:Thioredoxin-Disulfide Reductase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D013879:Thioredoxins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
chemical_list: D010168:Palmitates; D017382:Reactive Oxygen Species; C491203:TXN protein, human; D010171:Palmitoyl Coenzyme A; D013879:Thioredoxins; D013880:Thioredoxin-Disulfide Reductase; D005947:Glucose
keywords: Free fatty acids; Oxidative stress; Palmitoylation; Thioredoxin; Thioredoxin reductase