Identification of hepatic protein-protein interaction targets for betaine homocysteine S-methyltransferase

Abstract

Protein-protein interactions are an important mechanism for the regulation of enzyme function allowing metabolite channeling, crosstalk between pathways or the introduction of post-translational modifications. Therefore, a number of high-throughput studies have been carried out to shed light on the protein networks established under different pathophysiological settings. Surprisingly, this type of information is quite limited for enzymes of intermediary metabolism such as betaine homocysteine S-methyltransferase, despite its high hepatic abundancy and its role in homocysteine metabolism. Here, we have taken advantage of two approaches, affinity purification combined with mass spectrometry and yeast two-hybrid, to further uncover the array of interactions of betaine homocysteine S-methyltransferase in normal liver of Rattus norvegicus. A total of 131 non-redundant putative interaction targets were identified, out of which 20 were selected for further validation by coimmunoprecipitation. Interaction targets validated by two different methods include: S-methylmethionine homocysteine methyltransferase or betaine homocysteine methyltransferase 2, methionine adenosyltransferases α1 and α2, cAMP-dependent protein kinase catalytic subunit alpha, 4-hydroxyphenylpyruvic acid dioxygenase and aldolase b. Network analysis identified 122 nodes and 165 edges, as well as a limited number of KEGG pathways that comprise: the biosynthesis of amino acids, cysteine and methionine metabolism, the spliceosome and metabolic pathways. These results further expand the connections within the hepatic methionine cycle and suggest putative cross-talks with additional metabolic pathways that deserve additional research.

Protein-protein interactions involving enzymes of the mammalian methionine and homocysteine metabolism

“…BHMT targets identified in that study were consider validated when the interaction was verified by two independent methods [32]. This was the case of proteins belonging to the methionine cycle such as BHMT2, which was identified independently by both AP-MS and yeast-two hybrid [32], and previously found in other high-throughput studies [41,42] (Fig.…”

“…BHMT-Prkaca [32]). Other PTMs identified in methionine and Hcy metabolic enzymes are related to redox stress, a main mechanism involved in a large variety of pathologies and aging.…”

“…interaction [32,110]. Additionally, high-throughput studies aimed to establish the whole human interactome have also identified several putative BHMT interaction partners [41,42].…”

“…More recently, two complementary high-throughput methods were used for the identification of the BHMT interactome in rat liver [32]. On one hand, rat liver cytosol and AP-MS, utilizing three consecutive chitin-based affinity columns that differed in the proteins bound to the beads (none, intein and intein-BHMT).…”

“…tubulin). On the other, a parallel yeast two-hybrid screening of a rat liver cDNA library further enriched the list with 15 additional partners [32]. Analysis of these targets using STRING and KEGG allowed the classification of a limited subset of these proteins into four main pathways, namely: metabolic pathways (16), spliceosome (7), biosynthesis of amino acids (7) and cysteine and methionine metabolism (5).…”