Isoaspartate, Carbamoyl phosphate synthase-1, and carbonic anhydrase-III as biomarkers of liver injury
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 accu...
| Main Authors: | , , , , , , , , , , , , , , |
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| Format: | Article |
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Elsevier
2016
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| Online Access: | https://eprints.nottingham.ac.uk/35114/ |
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| author | 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 |
| author_facet | 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 |
| author_sort | Carter, Wayne |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | 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. |
| first_indexed | 2025-11-14T19:25:13Z |
| format | Article |
| id | nottingham-35114 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| last_indexed | 2025-11-14T19:25:13Z |
| publishDate | 2016 |
| publisher | Elsevier |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-351142020-05-04T17:42:03Z https://eprints.nottingham.ac.uk/35114/ 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 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. Elsevier 2016-03-13 Article PeerReviewed 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 and Kharbanda, Kusum (2016) Isoaspartate, Carbamoyl phosphate synthase-1, and carbonic anhydrase-III as biomarkers of liver injury. Biochemical and Biophysical Research Communications, 458 (3). pp. 626-631. ISSN 1090-2104 Alcohol-induced liver injury; Carbamoyl phosphate synthase-1; Carbonic anhydrase-III; Isoaspartate; Liver proteome; Protein isoaspartyl methyltransferase http://www.sciencedirect.com/science/article/pii/S0006291X15002314 doi:10.1016/j.bbrc.2015.01.158 doi:10.1016/j.bbrc.2015.01.158 |
| spellingShingle | Alcohol-induced liver injury; Carbamoyl phosphate synthase-1; Carbonic anhydrase-III; Isoaspartate; Liver proteome; Protein isoaspartyl methyltransferase 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 |
| title | Isoaspartate, Carbamoyl phosphate synthase-1, and carbonic anhydrase-III as biomarkers of liver injury |
| title_full | Isoaspartate, Carbamoyl phosphate synthase-1, and carbonic anhydrase-III as biomarkers of liver injury |
| title_fullStr | Isoaspartate, Carbamoyl phosphate synthase-1, and carbonic anhydrase-III as biomarkers of liver injury |
| title_full_unstemmed | Isoaspartate, Carbamoyl phosphate synthase-1, and carbonic anhydrase-III as biomarkers of liver injury |
| title_short | Isoaspartate, Carbamoyl phosphate synthase-1, and carbonic anhydrase-III as biomarkers of liver injury |
| title_sort | isoaspartate, carbamoyl phosphate synthase-1, and carbonic anhydrase-iii as biomarkers of liver injury |
| topic | Alcohol-induced liver injury; Carbamoyl phosphate synthase-1; Carbonic anhydrase-III; Isoaspartate; Liver proteome; Protein isoaspartyl methyltransferase |
| url | https://eprints.nottingham.ac.uk/35114/ https://eprints.nottingham.ac.uk/35114/ https://eprints.nottingham.ac.uk/35114/ |