title: Isoaspartate, carbamoyl phosphate synthase-1, and carbonic anhydrase-III as biomarkers of liver injury.
abstract: We had previously shown that alcohol consumption can induce cellular isoaspartate protein damage via an impairment of the activity of protein isoaspartyl methyltransferase (PIMT), an enzyme that triggers repair of isoaspartate protein damage. To further investigate the mechanism of isoaspartate accumulation, hepatocytes cultured from control or 4-week ethanol-fed rats were incubated in vitro with tubercidin or adenosine. Both these agents, known to elevate intracellular S-adenosylhomocysteine levels, increased cellular isoaspartate damage over that recorded following ethanol consumption in vivo. Increased isoaspartate damage was attenuated by treatment with betaine. To characterize isoaspartate-damaged proteins that accumulate after ethanol administration, rat liver cytosolic proteins were methylated using exogenous PIMT and (3)H-S-adenosylmethionine and proteins resolved by gel electrophoresis. Three major protein bands of ∼ 75-80 kDa, ∼ 95-100 kDa, and ∼ 155-160 kDa were identified by autoradiography. Column chromatography used to enrich isoaspartate-damaged proteins indicated that damaged proteins from ethanol-fed rats were similar to those that accrued in the livers of PIMT knockout (KO) mice. Carbamoyl phosphate synthase-1 (CPS-1) was partially purified and identified as the ∼ 160 kDa protein target of PIMT in ethanol-fed rats and in PIMT KO mice. Analysis of the liver proteome of 4-week ethanol-fed rats and PIMT KO mice demonstrated elevated cytosolic CPS-1 and betaine homocysteine S-methyltransferase-1 when compared to their respective controls, and a significant reduction of carbonic anhydrase-III (CA-III) evident only in ethanol-fed rats. Ethanol feeding of rats for 8 weeks resulted in a larger (∼ 2.3-fold) increase in CPS-1 levels compared to 4-week ethanol feeding indicating that CPS-1 accumulation correlated with the duration of ethanol consumption. Collectively, our results suggest that elevated isoaspartate and CPS-1, and reduced CA-III levels could serve as biomarkers of hepatocellular injury.
authors: School of Medicine, University of Nottingham, Royal Derby Hospital Centre, Derby, DE22 3DT, UK. Electronic address: wayne.carter@nottingham.ac.uk.
authors: Wayne G
authors: WG
authors: Carter
authors: School of Medicine, University of Nottingham, Royal Derby Hospital Centre, Derby, DE22 3DT, UK.
authors: Vasanthy
authors: V
authors: Vigneswara
authors: School of Medicine, University of Nottingham, Royal Derby Hospital Centre, Derby, DE22 3DT, UK.
authors: Anna
authors: A
authors: Newlaczyl
authors: School of Medicine, University of Nottingham, Royal Derby Hospital Centre, Derby, DE22 3DT, UK.
authors: Declan
authors: D
authors: Wayne
authors: School of Medicine, University of Nottingham, Royal Derby Hospital Centre, Derby, DE22 3DT, UK.
authors: Bilal
authors: B
authors: Ahmed
authors: School of Medicine, University of Nottingham, Royal Derby Hospital Centre, Derby, DE22 3DT, UK.
authors: Stephen
authors: S
authors: Saddington
authors: School of Medicine, University of Nottingham, Royal Derby Hospital Centre, Derby, DE22 3DT, UK.
authors: Charlotte
authors: C
authors: Brewer
authors: School of Medicine, University of Nottingham, Royal Derby Hospital Centre, Derby, DE22 3DT, UK.
authors: Nikhilesh
authors: N
authors: Raut
authors: School of Medicine, University of Nottingham, Royal Derby Hospital Centre, Derby, DE22 3DT, UK.
authors: Henry K
authors: HK
authors: Gerdes
authors: School of Medicine, University of Nottingham, Royal Derby Hospital Centre, Derby, DE22 3DT, UK; Department of Pharmacology, University of the Basque Country, and Centro de Investigación Biomédica en Red de Salud Mental, Spain.
authors: Amaia M
authors: AM
authors: Erdozain
authors: School of Life Sciences, University of Nottingham, Queen's Medical Centre, Nottingham, UK.
authors: David
authors: D
authors: Tooth
authors: School of Life Sciences, University of Nottingham, Queen's Medical Centre, Nottingham, UK.
authors: Edward L
authors: EL
authors: Bolt
authors: Research Service, Veterans Affairs Nebraska-Western Iowa Health Care System, Omaha, NE, USA; Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, USA; Department of Biochemistry, University of Nebraska Medical Center, Omaha, NE, USA.
authors: Natalie A
authors: NA
authors: Osna
authors: Research Service, Veterans Affairs Nebraska-Western Iowa Health Care System, Omaha, NE, USA; Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, USA; Department of Biochemistry, University of Nebraska Medical Center, Omaha, NE, USA.
authors: Dean J
authors: DJ
authors: Tuma
authors: Research Service, Veterans Affairs Nebraska-Western Iowa Health Care System, Omaha, NE, USA; Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, USA; Department of Biochemistry, University of Nebraska Medical Center, Omaha, NE, USA.
authors: Kusum K
authors: KK
authors: Kharbanda
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000032:analysis* / Q000378:metabolism; D024403:Carbonic Anhydrase III / Q000032:analysis* / Q000378:metabolism; D002478:Cells, Cultured; D056486:Chemical and Drug Induced Liver Injury / Q000209:etiology / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D000431:Ethanol / Q000009:adverse effects; D026581:Isoaspartic Acid / Q000032:analysis* / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012435:S-Adenosylhomocysteine / Q000378:metabolism
chemical_list: D015415:Biomarkers; D026581:Isoaspartic Acid; D000431:Ethanol; D012435:S-Adenosylhomocysteine; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; D024403:Carbonic Anhydrase III; D002222:Carbamoyl-Phosphate Synthase (Ammonia)
keywords: Alcohol-induced liver injury; Carbamoyl phosphate synthase-1; Carbonic anhydrase-III; Isoaspartate; Liver proteome; Protein isoaspartyl methyltransferase