TY - JOUR
T1 - Isoaspartate, carbamoyl phosphate synthase-1, and carbonic anhydrase-III as biomarkers of liver injury
AU - Carter, Wayne G.
AU - Vigneswara, Vasanthy
AU - Newlaczyl, Anna
AU - Wayne, Declan
AU - Ahmed, Bilal
AU - Saddington, Stephen
AU - Brewer, Charlotte
AU - Raut, Nikhilesh
AU - Gerdes, Henry K.
AU - Erdozain, Amaia M.
AU - Tooth, David
AU - Bolt, Edward L.
AU - Osna, Natalie A.
AU - Tuma, Dean J.
AU - Kharbanda, Kusum K.
N1 - Funding Information:
We are thankful to the National Institute on Alcohol Abuse and Alcoholism (NIAAA) ( http://www.niaaa.nih.gov/ ) R21 AA017296-A1 grant to WGC, DJT, and KKK to support this research. This work was also supported by Merit Review grant BX001155 to KKK from the Department of Veterans Affairs . Initial work on this project was also funded by a Wellcome Trust (UK) ( http://www.wellcome.ac.uk/index.htm ) Value In People (VIP) grant to WGC. AN and BA were supported by Wellcome Trust (UK) summer scholarship grants to WGC. SS was supported by a Physiological Society (UK) ( http://www.physoc.org/ ) summer scholarship grant to WGC. AME was funded by the Basque Government for work undertaken in the laboratory of WGC. The funders had no role in study design, data collection and analysis, preparation of the manuscript, or decision to publish.
Publisher Copyright:
© 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license.
PY - 2015/3/13
Y1 - 2015/3/13
N2 - 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 ∼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.
AB - 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 ∼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.
KW - Alcohol-induced liver injury
KW - Carbamoyl phosphate synthase-1
KW - Carbonic anhydrase-III
KW - Isoaspartate
KW - Liver proteome
KW - Protein isoaspartyl methyltransferase
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U2 - 10.1016/j.bbrc.2015.01.158
DO - 10.1016/j.bbrc.2015.01.158
M3 - Article
C2 - 25684186
AN - SCOPUS:84924019566
SN - 0006-291X
VL - 458
SP - 626
EP - 631
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 3
ER -