| PrefaceAldo-keto reductase family 1 B10(AKR1B10,also named aldose reductase-like-1, AKR1B10) identified from human hepatocellular carcinoma,is a novel member of aldo-keto reductase(AKR) superfamily with more than 70%of amino acid sequence identity to aldo-keto reductase family 1 B 1(AKR1B1,also known as aldose reductase, AR)。Unlike AKR1B1 with ubiquitous expression in various human tissues,AKR1B10 is primarily expressed in the small intestine and colon with a detectable level in the liver. However,AKR1B10 is highly upregulated in hepatocellular carcinoma,lung squamous cell carcinoma and lung adenocarcinoma in smokers,being a potential tumor marker for diagnosis and/or prognosis.AKR1B10 is a monomeric cytoplasmic protein with enzymatic activity toward carbonyls in the presence of NADPH.Recent studies have demonstrated that AKR1B10 expression significantly affects cell proliferation, clonogenic growth and susceptibility to acrolein and crotonaldehyde,indicating its potential role in protecting cells from carbonyl toxicity.However,its physiological function is still not clear.Carbonyls are electrophilic compounds,such as HNE,consistently produced intracellularly during metabolism of carbohydrates and lipids,and also known as important pathogens of GI diseases and tumors.Carbonyls widely exist in air,water, fruits,vegetables,meat,fish,and beverages.Especially,it produced intracellulary during oxygen extress.Due to their activity,carbonyls can react with free amino and sulfhydryl groups of proteins,peptides and amino acids,forming covalently modified adducts.These non-specific covalent modifications may lead to protein dysfunction, resistance to intracellular proteolysis,or depolymerization.Protein adducts may function as secondary messengers,autoantigens,or inhibitors of proteosomes,resulting in cellular damage and/or autoimmune disorders.Electrophilic carbonyls also bind nucleic acids(DNA),forming covalently modified DNA adducts.DNA adducts block DNA semiconservative replication performed by DNA polymerase and arrest transcription driven by RNA polymerase,leading to DNA mutations and breaks. Emerging evidence provided a considerable support that a pathogenic effect of carbonyl-derived DNA modifications might cause mutagenesis,carcinogenesis,and other age-related diseases.Therefore,dietary electrophilic carbonyls are regarded as important pathogens of GI diseases,including neoplasms.Via food consumption,GI cells that are repeatedly exposed to various reactive carbonyls.Long term and cumulative carbonyl exposure,even though minimal,may eventually induce a carcinogenic response in GI cells.For instance,exposure of F344 rats to 2, 4-hexadienal induced stomach hyperplasia,squamous papilloma,and carcinoma in rats, and high levels of malondialdehyde(MDA) in colonic mucosa was pathogenically related to neoplastic lesions in ulcerative colitis.In addition,local accumulation of acetaldehyde,microbially produced after alcohol consumption,has been considered a local carcinogenic factor for colon and gastric cancers.It is of great significance for public health to understand the host defensive mechanisms against carbonyls.This study demonstrates that AKR1B10 is an important enzyme for detoxifying dietary and cellular carbonyls and their glutathione conjugates.It further clarifies the pathogenesis mechanisms of cancer and may shed light on discovery of novel strategies for pronosis and treatment of cancer.Methods1.Construction of EGFP-C3/AKR1B10 according to gene homologous recombinant principle and Purification AKR1B10 protein and antibody.The full length human AKR1B10 cDNA was amplified from pBlu/AKR1B10 plasmid and the product was inserted into EGFP-C3 vector.The recombinant plasmid was identified by enzyme digestion.Purification of AKR1B10 and AKR1B1 proteins was prepared by using a pQE prokaryotic protein expression system for enzyme activity assay and AKR1B1 was used as a control.Highly specific AKR1B10 antibody was purified by SulfoLink Immobilization Kit.2.AKR1B10 enzyme activity assay.In kinetic assays with higher substrate concentrations,the decrease of NADPH was examined by a UV spectrophotometer at 340 nm to indicate enzyme activity,presented as oxidized NADPH (nmol)/min/mg protein.Under physiological conditions,human exposure to electrophilic carbonyls is generally low.To study the detoxicant role of AKR1B10 in a physiological status,we examined its enzymatic reactivity to the carbonyls at low concentrations by HPLC analysis of the enzymatic products.3.The protected effect of AKR1B10 on cells.Transfection efficiency indicated by EGFP expression in 293T cells was observed by fluorescence microscope. Expression of the EGFP-AKR1B10 fusion protein was verified by Western blot. Enzymatic activity of EGFP-AKR1B10 ectopically expressed in 293T cells was detected by using DL-glycealdehyde as a substrate.HNE toxicity in 293T transfected cells was detected by using a MTT assay.Intracellular metabolism of HNE was examined by HPLC analysis.Results1.EGFP-C3/AKR1B10 plasmid was successfully constructed,and AKR1B 10 and AKR1B1 protein were purified as well as the specific AKR1B 10 antibody.2.Kinetic parameters of AKR1B10 and AKR1B1 to carbonyls and glutathione conjugates:Km and kcat values at 110.1±12.2μM and 115.9±7.8 min-1 for acrolein,86.7±14.3μM and 103.4±3.5 min-1 for crotonaldehyde,30.9±7.1μM and 120.7±6.5 min-1 for 4-hydroxynonenal(HNE),60.6±18.5μM and 97.4±8.0 min-1for trans-2-hexanal and 95.7±5.6μM and 82.8±7.4 min-1 for trans-2, 4-hexadienal.Importantly,AKR1B10 exhibited enzymatic activity to crotonaldehyde at 0.90μM,HNE at 0.10μM,trans-2-hexanal at 0.10μM,and trans-2,4-hexadienal at 0.05μM.AKR1B10 also showed enzymatic activity to glutathione(GS)-carbonyl conjugates,except GS-HNE.3.An estimated transfection efficiency of approximately 85%-90%was obtained.This EGFP-AKR1B10 fusion protein(approximately 65.0 kDa,AKR 1B 10 plus EGFP) was detected by Western blot and yielded approximately a 4-fold increase of enzymatic activity to DL-glyceraldehyde,a common substrate for both AKR1B1 and AKR1B 10,indicating that ectopically expressed EGFP-AKR1B10 is functional.Cell viability tests revealed that the EGFP-AKR1B10 protein expressed in 293T cells significantly prevented the cytotoxicity of HNE compared to the vector control.4.To understand the protection mechanisms,we examined the intracellular metabolism of HNE treated at 1(subtoxic) or 5μM(toxic).The remaining HNE and its reduction product DHN was assessed by HPLC at indicated time points.In the 293T cells expressing the EGFP-AKR1B10 fusion protein,HNE was rapidly cleared up by AKR1B10-catalyzed reduction to DHN,which was detected within 30 seconds and then rapidly accumulated.The half-life of HNE at 5μM was approximately 1 1/2 min in EGFP-AKR1B10 expression 293T cells.These data suggest that AKR1B10 efficiently eliminates HNE in the 293T cells,but AKR1B1 did not.Conclusions1.The recombinant EGFP-C3 vector with AKR1B10 cDNA is easy to observe, effective to transfect and short to construct.His tag protein purification method can get high purity protein.Antibody for AKR1B 10 has a high specificity.2.AKR1B10 had markedly higher substrate affinity and product turnover rates as compared to AKR1B1.AKR1B10 can reduce the substrate at nM concentration suggesting that AKR1B10 is important for detoxifying cytotoxic carbonyls under physiological conditions.3.Both recombinant AKR1B1 and endogenous AKR1B10 have reduced effect on DL-glyceraldehyde,but cell viability tests revealed that the EGFP-AKR1B10 protein expressed in 293T cells significantly decreased the cytotoxicity of HNE as compared to AKR1B1.4.AKR1B10 efficiently eliminates HNE at toxic and subtoxic levels in transfected 293T cells as compared to AKR1B1.
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