title: Instability of C154Y variant of aldo-keto reductase 1C3.
abstract: Aldo-keto reductase (AKR) 1C3 is a cytosolic enzyme that metabolizes steroids, prostaglandins, toxic aldehydes and drugs. Recently, some nonsynonymous single nucleotide polymorphisms of AKR1C3 have been suggested to impact steroid and drug metabolism. In this study, we examined the effects of C154Y and L159V variants of AKR1C3 on stability and function of the enzyme. Both variants had been detected in patients with the neurodegenerative disease amyotrophic lateral sclerosis. Recombinant wild-type (WT), C154Y and L159V enzymes were similar in specific activity, but C154Y displayed much lower thermostability than WT and L159V. C154Y was inactivated by 10-min incubation at >25 °C, and about 90% of its activity was lost at 40 °C. Differential scanning fluorimetry revealed that Tm(thermal denaturation midpoint) of C154Y was lower than that of WT. In order to study the cause of thermosensitivity of C154Y, we prepared C154F and C154S mutant AKR1C3s. Like C154Y, C154F was highly sensitive to thermal inactivation, whereas C154S showed almost the same thermostability as WT. The C154F and C154Y variants induced secondary and tertiary structural changes in AKR1C3 at 40 °C as reflected by their altered circular dichroism and 8-anilinonaphthalene-1-sulfonate fluorescence characteristics. These results suggest that the replacement of C154 with a residue possessing a bulky aromatic side-chain impairs the folding of the α-helix containing C154 and its neighboring secondary structures, leading to low thermostability of AKR1C3. AKR1C3 metabolizes cytotoxic 4-oxo-2-nonenal into a less toxic metabolite, and overexpression of WT in HEK293 cells alleviated the 4-oxo-2-nonenal-induced cytotoxicity. In contrast, the overexpression of C154Y in the cells did not show such a significant protective effect, suggesting that C154Y is unstable in cells.
authors.affiliation: Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196, Japan. Electronic address: sendo@gifu-pu.ac.jp.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Endo
authors.affiliation: Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196, Japan.
authors.forename: Sayaka
authors.initials: S
authors.lastname: Takada
authors.affiliation: Department of Molecular Pathobiochemistry, Graduate School of Medicine, Gifu University, Gifu 501-1193, Japan.
authors.forename: Ryo P
authors.initials: RP
authors.lastname: Honda
authors.affiliation: Neurology Department, Ulm University, Ulm 89081, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Müller
authors.affiliation: Neurology Department, Ulm University, Ulm 89081, Germany.
authors.forename: Jochen H
authors.initials: JH
authors.lastname: Weishaupt
authors.affiliation: Department of Pharmacology and Clinical Neuroscience, Umeå University, Umeå SE-901 85, Sweden.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Andersen
authors.affiliation: Neurology Department, Ulm University, Ulm 89081, Germany.
authors.forename: Albert C
authors.initials: AC
authors.lastname: Ludolph
authors.affiliation: Life Science Research Center, Gifu University, Gifu 501-1193, Japan.
authors.forename: Yuji O
authors.initials: YO
authors.lastname: Kamatari
authors.affiliation: Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196, Japan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Matsunaga
authors.affiliation: United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu 501-1193, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Kuwata
authors.affiliation: Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.
authors.forename: Ossama
authors.initials: O
authors.lastname: El-Kabbani
authors.affiliation: Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Ikari
mesh_terms: D000447:Aldehydes / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D000074425:Aldo-Keto Reductase Family 1 Member C3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D005470:Fluorometry; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D020641:Polymorphism, Single Nucleotide; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013696:Temperature
chemical_list: C403894:4-oxo-2-nonenal; D000447:Aldehydes; D011994:Recombinant Proteins; C487341:AKR1C3 protein, human; D000074425:Aldo-Keto Reductase Family 1 Member C3
keywords: AKR1C3; Aldo-keto reductase; Amyotrophic lateral sclerosis; Gene variants; Thermostability