Methylthioadenosine (MTA) Regulates Liver Cells Proteome and Methylproteome: Implications in Liver Biology and Disease

Abstract

Methylthioadenosine phosphorylase (MTAP), a key enzyme in the adenine and methionine salvage pathways, catalyzes the hydrolysis of methylthioadenosine (MTA), a compound suggested to affect pivotal cellular processes in part through the regulation of protein methylation. MTAP is expressed in a wide range of cell types and tissues, and its deletion is common to cancer cells and in liver injury. The aim of this study was to investigate the proteome and methyl proteome alterations triggered by MTAP deficiency in liver cells to define novel regulatory mechanisms that may explain the pathogenic processes of liver diseases. iTRAQ analysis resulted in the identification of 216 differential proteins (p < 0.05) that suggest deregulation of cellular pathways as those mediated by ERK or NFκB. R-methyl proteome analysis led to the identification of 74 differentially methylated proteins between SK-Hep1 and SK-Hep1+ cells, including 116 new methylation sites. Restoring normal MTA levels in SK-Hep1+ cells parallels the specific methylation of 56 proteins, including KRT8, TGF, and CTF8A, which provides a novel regulatory mechanism of their activity with potential implications in carcinogenesis. Inhibition of RNA-binding proteins methylation is especially relevant upon accumulation of MTA. As an example, methylation of quaking protein in Arg(242) and Arg(256) in SK-Hep1+ cells may play a pivotal role in the regulation of its activity as indicated by the up-regulation of its target protein p27(kip1) The phenotype associated with a MTAP deficiency was further verified in the liver of MTAP± mice. Our data support that MTAP deficiency leads to MTA accumulation and deregulation of central cellular pathways, increasing proliferation and decreasing the susceptibility to chemotherapeutic drugs, which involves differential protein methylation. Data are available via ProteomeXchange with identifier PXD002957 (http://www.ebi.ac.uk/pride/archive/projects/PXD002957).

Fig. 1.

One-carbon metabolites and proteome analysis in SK-Hep1+ cells. (A) MTAP, MTA, SAMe, and SAH levels. HepG2 extracts were used as positive control. MTA was down-regulated in MTAP expressing cells while SAMe/SAH ratio remained unchanged. (B) Heat map representing the differential proteins resulting from SK-Hep1 and SK-Hep1+ iTRAQ comparison.

Fig. 2.

MTA prevents apoptosis and provides drug resistance in SK-Hep1 cells. (A) IPA network representing functional connections between up- and down-regulated proteins in SK-Hep1+ cells. Nodes in green and red correspond to down- and up-regulated proteins in SK-Hep1+, respectively. Noncolored nodes are proposed by IPA and suggest potential targets functionally coordinated with the differential proteins. APEX1 and TGM2 levels deregulation suggested a drug resistance condition in SK-Hep1 cells. (B) Cellular sensitivity to doxorubicin exposure. MTAP deletion provides increased resistance to doxorubicin-induced apoptosis. (C) Functional validation of hypothesis based on IPA network. NFκB activation, CYP2E1, and HuR overexpression in liver hepatoma cells lacking MTAP was demonstrated by Western blotting.

Fig. 3.

MTA increases the proliferation rate according to a mechanism dependent on activation of the MAPK pathway. (A) IPA network MTAP from differentially regulated proteins mainly involved in cell proliferation and organization such as KRT8 and 18 in SK-Hep1 cells. (B) ERK1/2 activation was assessed by Western blotting, showing phosphorylation on Thr²⁰²/Tyr²⁰⁴ residues. (C) A dose-dependent MTA activation of ERK by phosphorylation was further confirmed in AML12 cells. (D) Cell proliferation was significantly reduced in SK-Hep1+ cells expressing MTAP as evidenced by cell counting.

Fig. 4.

Cellular methylation profile was significantly reduced upon incubation with MTA. (A) Overall protein R methylation analysis in untreated and MTA-treated SK-Hep1 and Sk-Hep1+ cells that confirm a dose-dependent methylation reduction. (B) Table and Venn diagram representing the number of R-peptides identified in SK-Hep1 and SK-Hep1+ cells. (C) IPA network from differential R-methylated proteins. Nodes in gray correspond to mono-R proteins specifically methylated in SK-Hep1+. The functional analysis of the 56 proteins revealed that they are mainly involved in RNA processing, protein synthesis and several processes regulating cell fate.

Fig. 5.

Profiling arginine methylation of quaking protein. (A) MS/MS spectrum of mono-methylated R242 residue identified from SK-Hep1+ cells using the Me-R⁴-100 antibody. Y₁₅ ion corresponding to the methylated-R residue is highlighted. (B) The activation of p27^kip1, a target of QKI, was assessed by Western blotting suggesting that methylation might increase QKI-5 activity as no change on its steady-state levels were detected.

Fig. 6.

Liver sensitivity induced by CCl₄ injury in MTAP deficient mice. (A) Characterization of MTAP and MTA level was performed by Western blotting and HPLC analysis, respectively, and showed that a partial deletion of MTAP+-/(40% decrease) in liver mice lead to a twofold increase of MTA hepatic level. (B) In agreement, consumption of exogenous MTA was twofold slower in MTAP± hepatocytes than in WT counterparts. (C) Exposure to a sublethal dose of CCl₄ (1 μl/g) induced a more severe damage in MTAP-deficient livers as evidenced by the more extent necrotic area and the larger increase on serum transaminases. Impaired recovery capacity upon 48 h in WT littermates was also observed. Up-regulation of CYP2E1 RNA level may explain the increased sensitivity to CCl₄.

Fig. 7.

HuR and QKI-5 proteins are deregulated in MTAP± mice as in SK-Hep1 cells. (A) Western blotting analysis revealed a threefold increase of HuR in MTAP± liver mice. (B) HuR activation by accumulation of MTA was further confirmed in WT and MTAP-deficient hepatocytes upon HGF stimulation. Furthermore, PCNA and cyclin A, HuR targets, were up-regulated. (C) QKI steady-state levels were similar in WT and MTAP± livers. (D) R-methyl-protein-IP combined with WB analysis revealed QKI-5 hypermethylation, providing support to our findings in SK-Hep1+ cells containing normal MTA levels.