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Isoaspartate, Carbamoyl phosphate synthase-1, and carbonic anhydrase-III as biomarkers of liver injury

Carter, Wayne; Vigneswara, Vasanthy; Newlaczyl, Anna; Wayne, Declan; Ahmed, Bilal; Saddington, Stephen; Brewer, Charlotte; raut, Nikhilesh; Gerdes, Henry; Erdozain, Amaia; Tooth, David; Bolt, Edward; Osna, Natalie; Tuma, Dean; Kharbanda, Kusum

Isoaspartate, Carbamoyl phosphate synthase-1, and carbonic anhydrase-III as biomarkers of liver injury Thumbnail


Vasanthy Vigneswara

Anna Newlaczyl

Declan Wayne

Bilal Ahmed

Stephen Saddington

Charlotte Brewer

Nikhilesh raut

Henry Gerdes

Amaia Erdozain

David Tooth

Edward Bolt

Natalie Osna

Dean Tuma

Kusum Kharbanda


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 3H-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 ~160kDa 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.

Journal Article Type Article
Acceptance Date Feb 4, 2016
Publication Date Mar 13, 2016
Deposit Date Jul 19, 2016
Publicly Available Date Jul 19, 2016
Journal Biochemical and Biophysical Research Communications
Print ISSN 0006-291X
Electronic ISSN 1090-2104
Publisher Elsevier
Peer Reviewed Peer Reviewed
Volume 458
Issue 3
Keywords Alcohol-induced liver injury; Carbamoyl phosphate synthase-1; Carbonic anhydrase-III; Isoaspartate; Liver proteome; Protein isoaspartyl methyltransferase
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