Identification of protein-protein interactions involving enzymes of hepatic homocysteine metabolism.

Abstract

[Background] Homocysteine metabolism enzymes are very abundant in liver, but little is known about their putative protein-protein interactions and their role in the regulation of this metabolic pathway and other cellular processes. [Aim] Our work was designed to identify new protein interaction partners for betaine homocysteine S-methyltransferase (BHMT) and methionine adenosyltransferase (MAT) I/III. [Methods] Yeast two-hybrid screening of a rat liver library was performed using either BHMT or MAT1 as baits, as well as affinity purification coupled to mass spectrometry using intein-BHMT as bait. Interactions were confirmed by coimmunoprecipitation and pull-down. The putative role of interaction targets with unknown function was explored by expression silencing in a rat hepatoma cell line, followed by microarray expression analysis. [Results] Yeast two hybrid screenings revealed 4-hydroxyphenylpyruvic acid dioxygenase and aldolase b as interactions targets of BHMT, whereas the oncogene PDRG1 was found interacting with MAT1. In parallel, affinity purification showed 131 non-redundant putative interaction targets for BHMT, out of which 20 were used for validation with additional techniques. Coimmunoprecipitation was performed after cotransfection of BHMT or MAT1 with each of their putative interaction partners in different cell lines. Using this approach the interaction of BHMT with S-methylmethionine homocysteine methyltransferase (BHMT2), MAT1, MAT2, cAMP-dependent protein kinase catalytic subunit alpha, 4-hydroxyphenylpyruvic acid dioxygenase and aldolase b was confirmed, as well as that of PDRG1 and MAT1. Furthermore, pull-down experiments demonstrated the ability of PDRG1 to interact with both MAT1 and MAT2 and to displace the regulatory MAT subunit from the MAT II hetero-oligomer. Both, PDRG1-MAT1 and PDRG1-MAT2 interactions led to a reduction in S-adenosylmethionine synthesis in vitro. In transfected cells, the PDRG1-MAT1 complex could only be isolated from the nuclear compartment, and a correlation between PDRG1 overexpression and DNA hypomethylation was detected. A reduction in the levels of this epigenetic modification was also observed upon PDRG1 overexpression in cells with endogenous MAT2 expression. Silencing of PDRG1 expression in rat hepatoma cells changed their steady-state expression profile in microarray analysis towards down regulation of genes involved in tumor progression. Altogether, the new interaction partners identified were used for network analysis, resulting in the identification of a limited number of KEGG pathways, such as the biosynthesis of amino acids and the spliceosome. [Conclusions] A set of new protein-protein interactions for BHMT and MAT proteins has been identified that expands the current knowledge of links of homocysteine metabolism, suggesting the existence of cross-talks with important metabolic routes, molecular machines, epigenetic modifications and tumor progression.