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

Francisco Garrido,María Ángeles Pajares,Roberto Velasco-García,Rocío Vargas-Martínez,María Pacheco,Horacio Serrano,Inmaculada Jorge,Jesús Vázquez,Francisco Portillo

doi:10.1371/journal.pone.0199472

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.

Highlights

High levels of homocysteine (Hcy) in plasma have been associated with a variety of pathologies that expand from cardiovascular disease to hearing loss [1,2,3,4]
The liver, and precisely, impairments in hepatic methionine metabolism seem to be responsible of these increased plasmatic Hcy concentrations, since approximately 50% of the ingested methionine is used in this organ [5]
The association of hyperhomocysteinemia (HHcy) with a large variety of diseases has increased the interest in the regulation of enzymes involved in Hcy metabolism and, among them betaine homocysteine S-methyltransferase (BHMT)

Summary

Introduction

High levels of homocysteine (Hcy) in plasma have been associated with a variety of pathologies that expand from cardiovascular disease to hearing loss [1,2,3,4]. When there is a need of methionine, Hcy can be methylated for the synthesis of this amino acid. The diet is the main source of betaine [8], but this metabolite can be obtained by choline oxidation in the mitochondria [9]. This oxidation allows recovery of one out of the three methyl groups donated by S-adenosylmethionine for the synthesis of phosphatidylcholine from phosphatidylethanolamine in one of the hepatic processes that more S-adenosylmethionine consumes [10]. BHMT becomes a link between osmoregulation, phospholipid synthesis and methionine/Hcy metabolism

Methods

Results

Conclusion